1. INTRODUCTION:

As concerns about environmental pollution, ecosystem equilibrium, health, and well-being have been increasing, interest in environment-friendly food, clothing, and shelter and healthy lives has been increasing, and the diffusion of well-being trends that meets consumers' demands to lead healthy lives is leading to increases in the consumption of diverse pollution-free and environment friendly foods and well-being foods that meet the consumers’ needs as such every year.¹

In line with changes in consumers’ lifestyles, new products related to health are being developed and distributed and enzyme related markets are also growing thanks to the atmosphere as such.² However, the enzyme foods commonly called by general consumers are sugar preserved food and correspond to a sort of 'syrup.' Therefore, this study aims to develop enzyme products with live enzyme activity through cereal grain fermentation and approximately mix extracts of foods helpful to modern people’s health and their diet, in which they are interested, with the enzyme products thereby developing new health supplement products to promote modern people’s healthy and happy lives following the extension of their average life span.

2. MATERIALS AND METHODS:

2.1. Experimental Material:

The Aspergillus kawachi used in this study was purchased from Suwon Jongkuk, and the grains were purchased on the market. Since the activities of protease and lipase in rice malt induced by Aspergillus kawachi fell short expectations, Asp. oryzae were fermented in soybeans to produce protease and lipase. If fermentation products with enzyme activity per se are pulverized and used as products, the activity will be lower than the target value and the products cannot be easily standardized. Therefore, the coenzyme was separated from the fermentation products and concentrated. Thereafter, the coenzyme concentrate was appropriately added to existing fermentation products and adjusted with a view to achieving the standardization of the product. Diverse natural materials were searched based on literature surveys and among them, corn silk, trifoliate orange, Job's-tears, and shiitake were selected considering taste, activity, and consumer awareness. The Job's-tears, trifoliate orange, corn silk, and shiitake were sufficiently dried at 50℃ for 3 days. After drying, they were pulverized and sieved with 100 mesh sieves, and the powder that passed the sieves was used as a sample.

2.1.1. Fermentation for Enzyme Production:

After putting 10 g of the malt sample into a 250 ml Erlenmeyer flask, 100 ml of a 0.5% saline solution was added, and the mixture was stirred slowly at 25°C for 3 hours to extract enzyme components. The extract was centrifuged at 10,000 g for 20 minutes and the supernatant was used as a coenzyme solution for experiments.

Seed malt manufacturing:

White rice was steamed for 30 minutes at 110℃ to make hard-boiled rice and the hard-boiled rice was grated well to homogenize the rice so that there was no lump. When the temperature of the rice became 32℃, Aspergillus kawachi and Aspergillus oryzae were inoculated to be 0.2% (w/w) and mixed to homogenize the mixture and the mixture was cultured. The mixture was overturned 18 hours later for the first time and 5 hours thereafter for the second time and the koji was finished 48 hours later when spores were sufficiently formed. The sample was widely spread out aseptically and thoroughly dried at 40 °C for two days. Thereafter, the same amount of dry heat sterilized starch was added and mixed thoroughly. The mixture was sieved using a sterilized 100mesh sieve to collect the starch containing the fungus spores and the collected starch was used as seed malt.

Malt Manufacturing:

White rice was washed and immersed in tap water for 2 hours, steamed thereafter and cooled to 30°C. Aspergillus kawachi seed malt prepared in advance was inoculated into the grains to become 0.5% of the grains and the grains were incubated at 28℃ for 5 days while diverse levels of humidity were maintained. Enzyme activity was measured at intervals of 12 hours.

Preparation of Enzyme Solution:

After putting 10 g of the malt sample into a 250 ml Erlenmeyer flask, 100 ml of 0.5% saline solution was added thereto, and the sample was slowly stirred at 25° C for 3 hours to extract enzyme components. The extract was centrifuged at 10,000 g for 20 minutes, and the supernatant was used as a coenzyme solution for experiments.

Measurement of α-amylase activity:

The measurement of α-amylase activity was carried out in accordance with the test method for enzyme foods under the Korean Food Standards Codex. After putting 5 ㎖ of 1% soluble starch solution, 13 ㎖ of Mcllvaine buffer solution (pH 7.0), and 1 ㎖ of 0.1% NaCl solution into a test tube, the mixture was heated to 37 ° C, and 1 ㎖ of the coenzyme solution was added thereto to induce reaction at 37°C for 20 minutes. After the reaction, the mixture was heated for 10 minutes at100 ° C to deactivate the enzyme, cooled to room temperature, and centrifuged at 10,000 rpm for 10 minutes to measure the quantity of reducing sugar in the supernatant.

Measurement of protease activity:

After putting 1 mL of the enzyme solution to 5 mL of the substrate (pH 7.0) made by dissolving 0.6% casein in 0.2 M phosphate buffer to induce reaction in a 37 ° C water bath for 10 minutes, the reaction was stopped by adding 5 mL of 0.44 M Trichloroacetic acid (TCA). After allowing the solution to stand for 30 minutes at room temperature, the solution was filtered (Whatman No.2) and 2 mL of filtrate was taken. Thereafter, 5 mL of 0.55 M Na2CO3 solution was added to the filtrate, 1 mL of folin reagent was added to develop color for 30 minutes, and the absorbance was measured at 660 nm.

Measurement of lipase activity:

The lipase activity was measured according to the method of Winkler and Stuckman (3) using p-nitrophenyl palmitate as a substrate. The p-nitrophenyl palmitate was dissolved in acetonitrile to become 10 mM. Thereafter, 10㎕ of the substrate solution, 40㎕ of alsolute ethanol, and 5～10㎕ of enzyme solution were added to 950 μl of 0.1 M Tris-HCl (pH 7.5) buffer solution, the solution was allowed stand for 30 minutes, and the amount of liberated p-nitrophenol was then measured by absorbance at 410 nm. The lipase activity (unit/g) was calculated by converting the number of ㎍ of p-nitrophenol generated when 1 g of malt reacted for 1 min.

2.1.2. Fermentation of soy beans using Asp. Oryzae:

Although proteases production using Aspergillus oryzae is generally achieved by fermenting steamed soybeans as with soybean lump fermentation, ³ this method was against the shortening of the fermentation period for productivity improvement, which is one of the purposes of this study, in that the fermentation period required is one month to several months in this case. In this study, the method of Hwang was modified to investigate optimal fermentation conditions that would enable proteolytic enzyme production within a short period of time.

-Manufacturing of seed malt

The Aspergillus oryzae for soybean fermentation was purchased from Suwon Jongkuk and the grains were purchased on the market.

- Fermentation of soybeans

Soybean powder made by steaming, drying, and roughly pulverize soybeans was adjusted to have a water content of 50% and the prepared Aspergillus oryzae was inoculated at a ratio of 0.5% (w/w). Thereafter, while the fermentation progressed at 28℃ for one week, the soybean powder was overturned at intervals of 24 hours and the α-Amylase, protease, Lipase activities in the fermentation product were measured.

2.1.3. Coenzyme separation from fermentation products and enrichment:

When the fermentation product with enzyme activity per se is pulverized into a product, the activity is lower than the target value and the standardization of the product is difficult.⁴ Therefore, the coenzyme was separated from fermentation product and concentrated and the coenzyme concentrate was appropriately added to the existing fermentation product and adjusted with a view to achieving the standardization of the product. The soybean fermentation product was used for enrichment of the coenzyme. Since the soybean fermentation product has relatively higher α-Amylase enzyme activity than rice malt and shows lipase activity, which did not appear in rice malt, if it is enriched to enhance enzyme inactivity, it may be applied to the standardization of the enzyme activity of overall products during enzyme product manufacturing processes. Sterilized water at 4℃ in the quantity corresponding to 20 times the weight of the soybean fermentation product was added to the soybean fermentation product and the solution was slowly stirred at 4℃ for 3 hours to extract soluble components containing enzymes. Thereafter, the solution was allowed to stand at 4°C for 24 hours to precipitate insoluble materials and remove the insoluble materials. Thereafter, the solution was centrifuged for 30 minutes at 10,000g and only supernatant was taken.

2.2. Statistical Analysis:

2.2.1. Analysis of antioxidant activity of natural substances:

Although there are studies regarding the antioxidant activity of corn silk, Job's-tears, shiitake and trifoliate orange⁵, no diet product made by adding corn silk, Job's-tears, shiitake and trifoliate orang after developing the optimal mix ratios of them was found yet in current market surveys.⁶ Job's-tears, trifoliate orange, corn silk, and shiitake were dried sufficiently for 3 days at 50°C. After drying, they were dried, pulverized and sieved using 100 mesh sieves and the powder that passed the sieve was used as samples.

To analyze the antioxidant activity of the Job's-tears, trifoliate orange, corn silk and shiitake samples used, the total phenol contents, total flavonoid contents, and DPPH radical scavenging rates were measured. When the total phenol contents were compared, the total phenol content of Job's-tears extract was shown to be 14㎍/g, that of trifoliate orange extract was shown to be 310㎍/g, that of corn silk extract was shown to be 43㎍/g, and that of shiitake extract was shown to be 34㎍/g. Therefore, trifoliate orange extract showed the highest total phenol content followed by corn silk extract, shiitake extract and Job's-tears extract in order of precedence (Table 1).

Table 1. Composition of the enzyme diet product

Ingredient	Content (%)
Powder of fermented grains	20
Powder of fermented soybeans	10
Trifoliate orange powder	5
Corn silk powder	10
Job’s-tears powder	15
Shiitake powder	10
Wheat flour	20
Lactic acid bacteria (bifidus)	10
Total	100

When total flavonoid contents were compared, the total flavonoid contents of Job’s-tears extract, trifoliate orange extract, corn silk extract, and shiitake extract were shown to be 10㎍/g, 570㎍/g, 65㎍/g, and 20㎍/g, respectively. Therefore, trifoliate orange extract showed the highest total flavonoid content followed by corn silk extract, shiitake extract, and Job’s-tears extract in order of precedence. The overall antioxidant activity of the samples used was lower than the results of other researchers.⁷ This is thought to be attributable to various conditions such as sample harvesting time and the soil where the samples were cultivated. However, the fact that the antioxidant activity of trifoliate orange was shown to be generally much higher compared to the other samples was noteworthy. The antioxidant activities of antioxidant natural substances used in this study were analyzed and in the results, trifoliate orange powder extract showed the highest antioxidant activity followed by corn silk extract, shiitake extract, and Job's-tears extract in order of precedence.

2.2.2. Development of test product of enzyme products helpful to diet:

A test product was developed by maxing trifoliate orange, corn silk, Job’s-tears, and shiitake and adding wheat flour and lactic acid bacteria (bifidus 10¹⁰/g) and the enzyme activity, antioxidant activity, and aflatoxin of the developed product were analyzed.

2.2.3. Analysis of the nutritive components of the developed product:

Nine nutritive components including crude protein of the developed product were analyzed. The calorie of the product was 405kcal/100g and the contents of carbohydrate, crude protein, and crude fat were 43g(43%), 42g(42%) and 4g(4%) per 100g respectively. When the offered amount per time was assumed to be 50g, the nutritive component contents and daily nutrient reference value ratios according to offered amounts were as shown in (Table 2).

Table 2. Nutritive component contents and daily nutrient reference value ratios according to offered amounts per time of the developed product

Nutritive component Offered amount per time (30g)
	Contents in offered amount per time	% nutrient reference value
Calorie	122 kcal
Carbohydrate	15 g	5 %
Sugars	0
Protein	13 g	23 %
Fat	1 g
Saturated fat	0	2 %
Trans fat	0
Cholesterol	7 ㎎	2 %
Natrium	0 ㎎	0 %

[Note] % nutrient reference value: ratio to daily nutrient reference value

3. RESULTS AND DISCUSSION:

To analyze the antioxidant activity levels of the Job’s-tears, trifoliate orange, corn silk, and shiitake samples used in this study, the total phenol contents, total flavonoid contents, and DPPH radical scavenging rates were measured. When total phenol contents were compared, the total phenol content of Job’s-tears extract was shown to be 14㎍/g, that of trifoliate orange extract was shown to be 310㎍/g, that of corn silk extract was shown to be 43㎍/g, and that of shiitake extract was shown to be 34㎍/g. Therefore, trifoliate orange extract showed the highest total phenol content followed by corn silk extract, shiitake extract, and Job’s-tears extract respectively (Table 3). When total flavonoid contents were compared, the total flavonoid contents of Job’s-tears extract, trifoliate orange extract, corn silk extract, and shiitake extract were shown to be 10㎍/g, 570㎍/g, 65㎍/g, and 20㎍/g, respectively. Therefore, trifoliate orange extract showed the highest total flavonoid content followed by corn silk extract, shiitake extract, and Job’s-tears extract in order of precedence. Although there are studies regarding the antioxidant activity of corn silk, Job's-tears, shiitake and trifoliate orange, no diet product made by adding corn silk, Job's-tears, shiitake and trifoliate orang after developing the optimal mix ratios of them was found yet in current market surveys^8’. In this study, corn silk, Job's-tears, shiitake and trifoliate orange were appropriately added to the developed enzyme product with a view to developing enzyme products with obesity prevention and antioxidant effects.

Table 3. Analysis of antioxidant activity of Job’s-tears, trifoliate orange, corn silk, and shiitake extracts

Division	Job’s-tears	trifoliate orange	Corn silk	shiitake
Total phenol content(㎍/g)	14	310	43	34
Total flavonoid content	10	570	65	20
DPPH radical scavenging rates of 10% extracts (%)	22	95	65	87

4. CONCLUSION:

Obesity, a state where excess fat has been accumulated in the body due to unbalanced energy intake and consumption, is becoming a serious social problem worldwide,⁹ and has obesity has been identified as a major cause of geriatric diseases such as diabetes, hypertension, cardiovascular diseases and hyperlipidemia, the World Health Organization (WHO) has designated obesity as a major health care problem since 2000. ¹⁰ Therefore, since we have developed an enzyme product which has live enzyme activity through cereal grain fermentation with natural substances having anti-obesity function and antioxidant effect while having little side effects, the enzyme product will not only provide obesity prevention and improvement effects but also help the prevention and relief of diseases due to oxidative stress in addition to antiaging.

5. ACKNOWLEDGMENT:

This study was conducted with the support of the 2018 university research fund of Gwangju University.

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Received on 12.06.2019 Modified on 18.07.2019

Research J. Pharm. and Tech. 2019; 12(10):5042-5046.

DOI: 10.5958/0974-360X.2019.00875.8