Characteristics of fermented seasoning sauces using Tenebrio molitor larvae

Accepted Manuscript Characteristics of fermented seasoning sauces using Tenebrio molitor larvae

Joo-Hyoung Cho, Hui-Ling Zhao, Ji-Su Kim, Soo-Hee Kim, Chang-Ho Chung PII: DOI: Reference:

S1466-8564(17)30510-6 doi:10.1016/j.ifset.2017.10.010 INNFOO 1871

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Innovative Food Science and Emerging Technologies

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Please cite this article as: Joo-Hyoung Cho, Hui-Ling Zhao, Ji-Su Kim, Soo-Hee Kim, Chang-Ho Chung , Characteristics of fermented seasoning sauces using Tenebrio molitor larvae. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Innfoo(2017), doi:10.1016/j.ifset.2017.10.010

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Ref. no.: INNFOO 1871

Characteristics of Fermented Seasoning Sauces using Tenebrio molitor Larvae

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Authors: Joo-Hyoung Cho, Hui-Ling Zhao, Ji-Su Kim, Soo-Hee Kim1 and Chang-Ho Chung*

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Affiliation:

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Department of Culinary Science and Foodservice Management, Sejong University,

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209 Neungdong-ro, Gwangjin-gu, Seoul, 05006, Republic of Korea.

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1. Department of Culinary Arts, Kyungmin University,

*corresponding author

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Tel. +822-3408-3222

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545 Seobu-ro, Uijeongbu-si, Gyeonggi-do, 11618, Republic of Korea

Fax. +822-3408-4314

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Email: [email protected]

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Characteristics of Fermented Seasoning Sauces using Tenebrio molitor Larvae Abstract As edible insects have come to be used as a dietary source of protein, related studies have been carried out in various fields. A liquid fermented seasoning was prepared using Tenebrio molitor larvae by applying the soy sauce fermentation process. Meju, koji, and roasted rice flour were combined with a 23% brine to produce a sauce. Aspergillus oryzae and Bacillus licheniformis were used to

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ferment raw or defatted insect larvae, and the resulting material was used as an insect meju. The insect koji was prepared with defatted insects and A. oryzae. Six sauce samples, including soy sauce with

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two different ratios of ingredients (meju:koji:roasted rice flour = 6:2:2 and 8:1:1) were prepared with raw T. molitor larvae (raw insect sauce), defatted larvae (defatted insect sauce), and soy (soy sauce)

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after 20 days of fermentation at 25°C. No difference in the L value was observed among experimental groups, but the 6:2:2 ratio sauces generally had higher L values than did the 8:1:1 ratio sauces. The

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raw insect sauce showed the highest b value. The overall color change (ΔE) over time was not significant, but the value was high for the insect sauce. Browning tended to increase as fermentation

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continued in the soy and raw insect sauces. Browning of defatted insect sauce increased, but decreased sharply on day 20. Sample pH values were 5.70–6.19 on day 0 and 5.66–6.02 on day 20. No significant change in pH was observed among samples. Titratable acidity increased overall during

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fermentation. The 6:2:2 ratio sauces had higher total soluble solids, reducing sugars, and total sugars than did the 8:1:1 ratio sauces. Total nitrogen content of the sauces was 1.06–1.19% after 20 days of

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fermentation. The 6:2:2 ratio sauces showed greater amino-nitrogen content (0.26–0.32% on day 20) than did the 8:1:1 ratio sauces, indicating more efficient protein degradation. The percentage nitrogen degradation rates were higher for the insect sauces than for the soy sauce. Essential and nonessential

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amino acids, as well as amino acid derivatives, increased by 1.5–2 times during fermentation. The raw M6 insect sauce had the highest total free amino-acid content of 510.42 mg/kg. Glutamic acid, alanine,

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aspartic acid, serine, isoleucine, lysine, phenylalanine, and valine contents were high in the sauces.

Keywords: Tenebrio molitor larvae, fermented sauce, soy sauce, Ganjang, soybean

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1. Introduction

Protein is one of the most important constituents of the human body, and protein intake occurs mainly through meat. However, those in Asian countries, which have historically agricultural societies, have difficulty consuming meat because animals are used for farming or are expensive. They thus supplement protein intake through foods such as soybean, which is rich in protein and relatively easy

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to cultivate. As a typical soy food along with tofu, soy sauce is used as a seasoning for various foods and is one of the main protein sources in the Asian diet (Kwon, 1994; Jang, Woo & Lee, 2003). In

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brief, Asian soy sauce is produced from steamed soybeans as the major material and inoculated with Aspergillus species, such as A. oryzae or A. sojae, or naturally fermented by environmental

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microorganisms including fungi such as Aspegillus or Rhizopus and bacteria such as Bacillus and aged in high-concentration brine for 6 months to 1–2 years (Ryu, Cho, Chae & Park, 1993; Lee & Lee,

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1976). The former is a typical in Japan, and the latter is an example of Korean. As for the Korean, the traditional type is made by naturally fermented soybeans in brick form, called meju, and dipping them

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in salt water. The soybean protein is decomposed into various oligopeptides and free amino acids during fermentation and aging. Some amino acids are taste compounds in soy sauce. For example, alanine, glycine, serine, and lysine produce sweetness; histidine, arginine, and methionine are bitter in

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flavor; and glutamate contributes to umami taste. These free amino acids play important roles in the flavor of soy sauce (Nelson et al., 2002; Seo & Lee, 1992; Choi, Lee & Kim, 2016). This flavor is

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represented mainly by various volatile components, such as carbonyls, esters, alcohols, phenols, acids, sulfur compounds, and nitrogen compounds, and the main flavor substances are 4-hydroxy-2-ethyl-5methyl-3(2H)-furanone and 4-hydroxy-5-ethyl-2-methyl-3 (2H)-furanone (Nunomura, Sasaki, Asao &

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Yokotsuka, 1976). The various flavors of soy sauce differ depending on the raw materials and manufacturing method used (Seo, Hwang, Yang & Lee, 1995). Soybean is the most economical

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protein-rich vegetable crop, and its production is increasing continuously in the USA, Brazil, Argentina, and China. The import demand in other countries is increasing, but soy production is fluctuating (Lee, 2014). Although soybeans are a nutritionally superior plant protein, the disadvantages of soybean cultivation have been pointed out: depletion of forest land due to increased arable land, the more need for water, and increased use of insecticides and herbicides. About 2 billion people globally still suffer from food shortages. The demand for agricultural products is expected to increase 50% by 2030, and the food shortage problem is expected to become more serious because of increases in the world population (Wheeler & Von Braun, 2013; Keyzer, Merbis, Pavel & van Wesenbeeck, 2005). Insects are starting to attract attention as a way to solve the 3

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international food shortage problem, particularly as a potential protein source. Insects are consumed as an alternative source of nutrients in Africa, Latin America, Asia, and other parts of the world, particularly in rural areas, to overcome protein-energy malnutrition (Partha & Spandita, 2014). Insects are generally rich (50–60%) in protein and more productive, and their production generates less greenhouse gas than does that of animal meat; the global warming index is significantly lower for insect than for meat production. Insects are being watched as a food for the future (Bukkens, 1997;

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Oonincx & de Boer, 2012). In the future, insects can be used as alternative raw materials for foods that meet protein requirements, such as fish, shellfish, and soybeans, as well as meat. In order for insect proteins to be used as animal feed or edible food, economically viable mass production should

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be secured first, and technologies for processing and process automation should be developed to make

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insect industry economically competitive with the production of meat (van Huis, A. 2013). Except for a few countries, general consumers still have insufficient experience with edible insects

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as a raw material for food due to the dislike of insects, and acceptance of consumers remains low. Therefore, rather than ingesting such insects as they are, they are usually used in a form that does not appear as insects, that is, crushed or in powdered form and added to food (Sánchez-Muros, Barroso &

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Manzano-Agugliaro, 2014).

In particular, protein-degraded fermented food has been produced from insects using

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microorganisms in Wakayama Prefecture, Japan (The japan times, 2016), and grasshopper garments have been manufactured according to a fish sauce production method in a Nordic food laboratory in

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Denmark (Sloan, Legrand & Hindley, 2015). In addition, a study in China has been conducted using Tenebrio molitor larvae. Although experimental design approaches have been limited, these trials have suggested the possibility of using insect protein fermented products (Li & Li, 2008). Soy sauce

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manufacturing methods and characteristics differ among countries, and the progress of microbial fermentation studies of insect protein has been limited. Dried and powdered T. molitor larvae have

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50–52% protein and have passed all Korean Food and Drug Administration regulations for use as a food ingredient (Kim & Jung, 2013). Thus, T. molitor larvae are expected to be used in various foods in the near future.

This study was performed to develop a fermented insect sauce using T. molitor larvae. A. oryzae and Bacillus licheniformis, isolated from a soybean product fermented in Chungkukjang, Korea, were applied to degrade protein and produce the seasoned liquid. The characteristics of the products were compared with those of soy sauce (Jung, No & Kim, 2006; Tantimavanich, Pantuwatana, Bhumiratana & Panbangred, 1998).

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2. Materials and Methods Microwave-dried T. molitor larvae provided by the Yangju Agricultural Technology Center (Yangju-si, Gyeonggi-do, Korea) were used in this experiment. Soybeans grown in Jecheon, Chungcheongbuk-do, in 2016 were purchased from a local market in Hwayang-dong, Gwangjin-gu, Seoul, Korea. A commercially refined salt (>99% pure; Hanju Co., Ulsan, Korea) was used for the experiment. Proximate analysis of raw materials is shown in Table 1.

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B. licheniformis (KACC 15829) was purchased from the Korean Agricultural Culture Collection (Jeonju, Jeollabuk-do, Korea) and A. oryzae was purchased from Chungmu Fermentation Co., Ltd.

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(Ulju, Ulsan, Gyungsangnam-do, Korea). B. licheniformis was inoculated into sterile tryptic soy broth (Becton-Dickinson and Co., Brea, CA, USA) and incubated at 37°C for 24 h, and absorbance was

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measured at 600 nm using an ultraviolet spectrophotometer (Spectronic 20D+; Milton Roy Co., Pacisa, Madrid, Spain) The culture was diluted until optical density reached 0.3, and then used to

Meju, koji, and liquid sauce preparation

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produce meju. In addition, A. oryzae was used to prepare koji and meju in powdered form.

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The soy sauce was prepared by referring to methods reported in previous research (Jeong, Shin, Jeong, Yang & Jeong, 2014; Lee et al., 2002). Briefly, 700 g soybeans was washed and immersed three times in distilled water at room temperature for 18 h. Then, the shells were removed and the

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soybeans were air dried. The dehulled soybeans (660 g) were used as the control. Raw and defatted. T.

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molitor larvae were prepared. The defatted type was pressed using a home mill–type-press (HD-333; Ggaebaksa. Co., Seoul, Korea). The same amount of larvae as raw dehulled soybean dry weight was used. All samples were autoclaved at 121°C for 30 min and cooled before use.

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Then, 1% (v/w) overnight-grown B. licheniformis (absorbance of 0.3 at 600 nm) and 0.5% (w/w) A. oryzae spores were inoculated on soybean and larvae and fermented in an incubator at 30°C for 38 h,

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then dried at 60°C for 24 h. The resulting products with soybean and larvae were called soybean and insect meju, respectively. Defatted T. molitor larvae powder was used to prepare the koji. After adding water to the powder to 42.1% moisture, the mixture was autoclaved at 121℃ for 30 min. A. oryzae spores (0.5% [w/w]) were inoculated and the koji was prepared after fermentation in an incubator at 30°C for 48 h, then dried at 60°C until moisture reached 14%. Rice flour was roasted in a frying pan for about 10 min until golden brown. Soy (or insect) meju, koji, and roasted rice flour were mixed at ratios of 8:1:1 and 6:2:2 by weight, respectively, to prepare the sauces. Twice the weight of 23% saltwater to total weight of meju, koji, and roasted rice flour was added and fermented in an incubator at 25°C for 20 days. 5

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The liquid samples were filtered through kitchen-type filter paper and centrifuged at 8,000 rpm for 15 min (HMR-220IV; Hanil Industrial Co., Seoul, Korea) to remove fat and solids. The centrifuged liquid was used for the analysis. In addition, analytical samples collected on days of 0, 6, 13, and 20 were subjected to testing. For sensory evaluation, the separated liquid phase was boiled at 100℃ for 10 min and the suspension was removed and cooled to prepare the sauce samples. The labels and ratios of the samples are shown in Table 2. This study was carried out in duplicate, and the results are

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shown as the average of the results. Proximate analysis

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The moisture contents were determined after drying at 105°C. The micro-Kjeldahl method was employed to determine the crude protein (N × 6.25). Crude lipids were extracted with petroleum ether

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using a Soxhlet apparatus, and the ash content (gravimetric) was determined based on methods outlined in AOAC (AOAC, 2000). Total carbohydrates was calculated by the difference method;

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summing the values of moisture, crude protein, ash, and crude fat (ether extract) and subtracting the sum from 100 (McDonald, et al, 1973).

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Color

To measure chromaticity, 5 mL prepared sauce was collected and measured using a colorimeter (CR-300 Chroma Meter; Minolta, Tokyo, Japan). The measured values were determined by repeatedly

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measuring the lightness (L) value, indicating brightness; the a value, indicating redness; the b value,

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indicating yellowness; and the color change (ΔE) value. The standard plate used had L, a, and b values of 98.72, −0.34, and 1.20, respectively.

Browning

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ΔE: {(L-L’)2 + (a − a’) 2 + (b − b’)2}1/2

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Browning was measured by diluting the sample 10 times with distilled water and filtering through a 0.2 μm membrane filter (Whatman, Bedford, MA, USA) to remove solid matter, which could affect absorbance measurement. The solution was analyzed using a spectrophotometer (X-MA 1200V; Human Corp., Hunan, China). Absorbance was measured at 420 nm (Chung & Toyomizu, 1968). pH, titratable acidity, total soluble solids, and salinity pH was measured with a pH meter (Model PB-10; Sartorias, Goettingen, Germany), and titratable acidity was calculated by titrating 10 mL sample with 0.1 N NaOH to pH 8.3 (Lee et al., 2002). Total soluble solids were determined using a refractometer (PR-40DMF; Atago Co., Tokyo, Japan) based on placement of undiluted liquid portions of samples into microtubes for centrifugation at 10,000×g 6

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for 10 min (5415 C; Brinkmann Instruments Inc., Rockaway, NJ, USA). Salinity was measured by the Mohr method using 10 mL of a 40-fold diluted soy sauce solution. One milliliter of 10% (v/v) K 2CrO4 solution was added and the amount of 0.1 N AgNO3 solution consumed was measured by adding the solution until the reddish-brown color did not disappear for 15 s (Joo, Sohn & Park, 1997a).

Reducing sugar and total sugar contents

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Three milliliters of dinitrosalicylic acid reagent were added to 1 mL sample solution diluted 50 times with distilled water and heated in a water bath at 100°C for 10 min to measure reducing sugars (Miller, 1959). After cooling, absorbance was measured at 550 nm. Glucose (Sigma Co., St. Louis,

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MO, USA) was used for calibration at a concentration range of 0.2–1.0 mg/mL. Total sugar content

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was determined by the phenol-H2SO4 method (Dubois, Gilles, Hamilton, Rebers & Smith, 1956). A 0.1-mL aliquot of sauce was diluted with distilled water to 100 mL. One milliliter of the filtered

30 min, absorbance was measured at 470 nm.

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sample solution was mixed with 1 mL 5% phenol solution and 5 mL concentrated sulfuric acid. After

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Total nitrogen, amino-nitrogen, and the nitrogen degradation ratio Total nitrogen was measured by the Kjeldahl method. A 2-mL aliquot of the sauce sample was added to a Kjeldahl flask. After the addition of 25 mL H2SO4, it was digested with an accelerator

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(Kjeltabs-K2SO4 3.5 g, Se 3.5 mg; Gerhardt, Königswinter, Germany). After decomposition was

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completed, the product was distilled and neutralized in a distillation apparatus (Vapodest; Gerhardt) and titrated with 0.1 N H2SO4 to convert the total number of milliliters consumed to total nitrogen. Amino-nitrogen was measured by the formal method (Choi et al., 2007). Forty-fold diluted sauce

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was homogenized with a stomacher (Bagmixer 400, speed 7, 2 min; Interscience Co., Saint-Nom-laBretèche, France). In this experiment, 20 mL neutral formalin solution and 20 mL distilled water were added to 25 mL diluent and titrated to pH 8.4 with 0.1 N NaOH solution. A 40-mL aliquot of distilled

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water was added to 25 mL diluted solution and titrated to pH 8.4 with 0.1 N NaOH solution, and amino-nitrogen content was calculated by subtracting the blank test solution. The nitrogen degradation ratio (NDR) was calculated as the percentage of amino-nitrogen divided by total nitrogen. Free amino acids To analyze the amino acid content of the soy sauce samples, 0.1 mL original sample solution was diluted with ultrapure water to 10 mL, and then centrifuged at 10,000 rpm for 10 min (centrifuge 5415C; Brinkmann Instruments Inc., Westbury, NY, USA). After filtering (Whatman), the sample was analyzed with an amino acid analyzer (Sykam, Eresing, Germany). At this time, the wavelength was set to 440 nm and 570 nm. A cation separation column (150 × 4.6 mm, LCA K07/Li) was set at a 7

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temperature of 37–74°C. Flow rates were 0.45 mL/min for the buffer and 0.25 mL/min for the reagent. Quantification of each amino acid was performed by area analysis using amino acid standards. Sensory Forty-four students from S University were selected for the preference test. The purpose of the experiment, tasting sauces, and ingredients were explained to and recognized by the participants. Four items (color, taste, flavor, and overall preference) were evaluated using a 7-point scale (1, much

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dislike; 5, moderate; 7, like). The evaluation method using boiled bean-sprout broth was selected so that the evaluators could easily evaluate mild saltiness. Bean sprout broth was added to each sauce

water to rinse the mouth was provided between each sample test.

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Statistics

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sample to reduce salinity to 0.6%. Each sample was placed in an odorless disposable cup. Drinking

Data were obtained in triplicate and analyzed statistically using SPSS software (ver. 19.0 for

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Windows; SPSS Inc., Chicago, IL, USA). Multiple comparisons among the experimental groups were performed using Tukey’s honestly significant difference test and the rank test. P values < 0.05 were

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considered to be significant.

3. Results and Discussion

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Color and browning

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In general, brightness (L value) did not change, but redness (a value) values increased, over time. Yellowness (b value) of the soybean and defatted samples decreased, whereas that of the raw insect sauce increased (Table 3). On day 20 of fermentation, the lightness value of the raw insect sauce was higher than those of the other samples. Redness values were lowest in soybean and higher in defatted

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insect sauce. Redness of the T. molitor larvae increased when the larvae were pressed and heated. Redness values were generally higher in the 6:2:2 sauces than in the 8:1:1 sauces. The yellowness

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value was highest in the raw insect sauce, possibly due to the effect of insect fat during fermentation (Lee et al., 2009).

The ΔE value (Table 3), indicating the overall color change, was highest for the insect sauce and did not change over time. The sauces produced in this study were fermented in glass jars, and ΔE values were similar to those reported by Jeon et al. (2002), who analyzed different soy sauces manufactured in different types of container. Browning, as assessed by absorbance, is shown in Fig. 1. Browning of sauces during fermentation is a non-enzymatic reaction, due to the participation of amidori rearrangement compounds and 8

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Maillard reaction intermediates, such as melanoidins and reductones (Okuhara, Saeki & Sasaki, 1972). Browning of the soy and raw insect sauces tended to increase as fermentation continued. On the other hand, browning increased in the defatted insects, but then decreased sharply and unexpectedly on day 20. Previous studies have shown that the amino-nitrogen content is correlated with the brownness of soy sauce (Kang, Lee, Ko & Hwang, 2011). In this study, the defatted insect sauce had higher aminonitrogen content and higher browning values than did the soy-based sauces.

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pH and titratable acidity Sample pH values were 5.70–6.19 on day 0 and 5.66–6.02 on day 20. No significant change was

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observed among samples (Table 4). This result was similar to the results of Jeong et al. (2014), who showed that the pH of soy sauce mixed with rice bran did not change (5.82–6.01) after 2 weeks of

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fermentation. Titratable acidity increased rapidly from 0.03% at the beginning of fermentation to 0.10% on day 6, and continued to increase until day 13. On day 20, acidity values of S8, S6, and M6 were

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the highest, whereas those of DM6, DM8, and M8 had decreased. Overall, the titratable acidity of the insect sauce was greater than that of the soy sauce. Titratable acidity increased in all samples during

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fermentation, consistent with previous reports (Song & Lee, 2013; Lee & Kim, 2002), and results were generally similar to those for pH and acidity in traditional Korean soy sauce reported by Kim, Kim and Chung (1996), who compared soy sauces prepared by different manufacturing methods.

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Salinity and total soluble solids

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Salinity on day 20 was 20.03–21.06%, and average salinity was 20.78%, with no significant difference among samples (Table 4). Salinity was greater than that of the commercially brewed soy sauce, but lower than that of traditional Korean soy sauce (Kim, Kim & Chung, 1996). The salt

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concentration of the final product could be adjusted by reducing salt during subsequent processing. Total soluble solids in all samples increased from day 6 to day 13 and decreased from day 13 to day 20. This tendency was thought to be due to free sugars and/or free amino acids used for microbial

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growth during the latter period of fermentation (Seo & Lee, 1992). On day 20, the value was highest for S6 (42.0° Brix) and lowest for M8 (36.5° Brix). In addition, the 6:2:2 ratio sauces had more soluble solids than did the 8:1:1 sauces. Because more koji and rice flour were added to the 6:2:2 ratio sauces than to the 8:1:1 sauces, more total soluble solids were observed in the former, probably due to higher amylase and protease activity levels. Reducing sugars and total sugar contents Reducing sugar content gives sweetness, which is one of the most important sensory indicators, to a sauce (Lee et al., 2015). Reducing sugar content tended to increase during fermentation, except in S6 9

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(Fig. 2). The increase in reducing sugars early in fermentation occurs when starch is saccharified by α-amylase produced by A. oryzae (Seo & Lee, 1993). Among all sauces, S6 had the highest reducing sugar content. In addition, the 6:2:2 ratio sauces had greater reducing sugar content than did the 8:1:1 ratio sauces, and reducing sugar content was greater in the preparations with soy sauce than that in the raw insect and defatted insect sauces. Changes in total sugar contents (P < 0.05) are shown in Fig. 3. Total sugar content was 1–2% on day 0, increased sharply until day 6 of fermentation, and was

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maintained until day 13. Subsequently, total sugar contents tended to decrease in all samples. It was greatest in S6. Overall, total sugar content of the 6:2:2 ratio sauces was greater than that of the 8:1:1

in the 6:2:2 ratio sauces, which was degraded by amylases.

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sauces in each experimental group, which appeared to be due to the two-fold greater rice flour content

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Reducing sugar content in S6 decreased from days 13 to 20, and total carbohydrate content decreased during this period. The amount of sugar resulting from decomposition of starch by amylase

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increased until day 6, was maintained until day 13, and then decreased on day 20. Sugar produced during soy sauce fermentation is used as a carbon source by microorganisms and is converted to acids

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by fermentation (Joo, Sohn & Park, 1997a).

When reducing sugar and total sugar contents were compared ([reducing sugar content / total carbohydrate content] × 100), differences in content were similar among samples. The mean amount

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of reducing sugar was 40% of total sugar on day 20. Sugar content was greater in the soy sauce than in the insect sauce. However, the percentage of reducing sugar in total sugar contents from insect

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sauce (average, 42.8%) was greater than that in the soy sauce (S8, 33.7%; S6, 34.9%). Total nitrogen, amino-nitrogen, and nitrogen degradation ratio

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Total nitrogen is a major factor affecting the quality of soy sauce (Table 5). In general, goodquality soy sauce contains 1.0–1.65% total nitrogen, 45% of which is composed of free amino acids and 45% of which is composed of simple peptides (Luh, 1995). Total nitrogen content of the sauces

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was 0.68–0.98% on day 0, and remained unchanged from days 6 to 12 of fermentation (P < 0.05). Total nitrogen content was greater in the defatted insect sauce than in other samples on day 0, but reached 1.31%, which was the greatest percentage, in S6 on day 20 of fermentation. In addition, S8, DM6, and DM8 had higher nitrogen contents and the raw insect sauce had lower amino-nitrogen content. However, total nitrogen content was >0.7% in all samples, which satisfied the soy sauce standard recommended by the FAO/WHO Codex Alimentarius Commission (2004). Amino-nitrogen content, which is an indicator of the degree of ripening and quality of the sauce during fermentation and aging, increased because available protein and protease activity increased, resulting in high concentrations of low molecular peptides and free amino acids (Kwon et al., 2010). 10

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Amino-nitrogen content was 0.04–0.09% at the beginning of fermentation. No significant difference was observed during the 20-day period after an increase to 0.25–0.35%. In addition, the 6:2:2 ratio sauces had higher amino-nitrogen contents than did the 8:1:1 ratio sauces, suggesting that the former ratio promoted ripening of the liquid seasoning. According to Park, Sohn and Park (1996), the amino-nitrogen content was 0.29% when soy sauce was fermented for 90 days. The NDR is an indicator of how fermentation has progressed by strain (Suh, Ryu & Hur, 1983).

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On day 0, the NDR for DM6 was 10.61% and that for M8 was 5.51%, but no significant difference was observed among the experimental groups. On day 20 of fermentation, M6 had the highest NDR value of 28.59%, and that of S8 was 22.47%. Overall, NDR values were higher for the 6:2:2 ratio

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sauces than for the 8:1:1 ratio sauces.

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Free amino acids

Free amino acid contents of the sauces were analyzed (Table 6). Essential and nonessential amino

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acids, as well as amino acid derivatives, increased by 1.5–2 times during fermentation. Free amino acid contents increased in soy sauce, whereas free amino acids increased in the insect sauce, but

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arginine, which produces a bitter taste, decreased significantly and proline, which is sweet, tended to decrease slightly.

This study was conducted to analyze the composition of seasoning sauce during relatively short -

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term fermentation (20 days). Total free amino acids was 4707.18 ~ 6117.08mg / 100g. These levels

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were comparable to a reference reported 5.91% in 90 days and 6.88% in 2 years in the case of aged soy sauce (Joo, Sohn & Park, 1997b). Ten essential amino acids, nine non-essential amino acids, and 17 amino acid derivatives were detected on day 20. Total amino acid contents in the 8:1:1 ratio sauces, excluding amino acid derivatives, were 3079.50, 4294.60, and 4411.61 mg/100g for soy and defatted

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and raw insect sauces, respectively. The values for the 6:2:2 ratio sauces were 4250.03, 4294.60, and 5104.17 mg/100g for soy and defatted and raw insect sauces, respectively. In general, raw insect sauce

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had greater amino acid contents than did the other sauces. In particular, the 6:1:1 ratio sauces had more amino acids than did the 8:1:1 ratio sauces. M6 also had a higher content of amino acid derivatives. Glutamic acid, which is an umami-tasting amino acid, yielded the greatest content among amino acids, with a range of 504.26–698.95 mg/100g in all samples. Proline gives sweetness and umami richness, and its content was 401.16–645.20 mg/100g in the insect sauces (Cho et al., 2013). Alanine, aspartic acid, and serine were the major nonessential amino acids. Soy sauce contained a greater amount of arginine, which produces a bitter taste and is considered to deteriorate flavor when present in a large amount. However, only a very 11

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small amount of arginine was detected in the insect sauce, so the bitterness level was expected to be low (Otagiri, Nosho, Shinoda, Fukui & Okai, 1985). The other essential amino acid contents did not differ significantly among samples. Leucine is an essential amino acid that was detected in greater amounts in all samples. Isoleucine, lysine, phenylalanine, and valine were also present in high concentrations. Glutamic acid and aspartic acid have been reported to contribute to umami taste, and glycine, alanine, and lysine are known to be

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sweet. Valine, leucine, isoleucine, and phenylalanine are the main bitter amino acids in traditional Korean soy sauce (Kim & Kim, 1980). The major amino acids of traditional Korean and commercial

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koji-type brewed soy sauce are alanine, glutamic acid, leucine, and valine (Kim, Kim & Chung, 1996). Soy sauces from this study had similar trends for these major amino acids. Most of the insect sauces

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had similar amino acid compositions, except that proline was the most abundant amino acid, along with glutamic acid. Citrulline was the most abundant amino acid derivative. In particular, M6 had the

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highest content of citrulline (423.97 mg/100g). Citrulline is a precursor amino acid that produces arginine (Marini, 2012), which effectively increases arginine and nitric oxide concentrations in plasma, thereby lowering blood pressure through vasodilation (Mohrman, 1988). In addition, urea and

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ornithine occurred in relatively high concentrations in the insect sauces. β-aminoisobutyric acid induces white fat to transform to brown fat, which is likely to be burned more effectively (Roberts et

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al., 2014).

The percentage of essential amino acids in total amino acids was 52.9% for S6, 52.6% for S8, 46.6%

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for M6, 47.7% for M8, 49.0% for DM6 and 47.0% for DM8. In a study of Jeong, Shin, Jeong, Yang and Jeong (2014), the ratio of soy sauce made from soybean koji (9: 1) with the highest amino acid content was 43.2% when fermented until day 14. In a study by Jeon, Sohn, Chae, Park and Jeon

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(2002), the traditional Korean type of soy sauce (60 day fermentation) has 51.4%, and soy sauce made using koji type was 47.9%, which was similar to the insect sauce of this study. In a study of Kim and

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Lee (2008), the percentage of soy sauce was 63.0% in 120 days of soy sauce fermentation. Recently, Zhao, Vázquez-Gutiérrez, Johansson, Landberg and Langton (2016) examined extraction of the larvae and found the protein extract was a good source of essential amino acids. The percentage of essential amino acidsin the study was 47.78%, similar to this study. Foods from soybean have high digestibility and high protein quality among vegetables. However, in the case of vegetable proteins, nutrient imbalances due to limited amino acids should be supplemented with relatively expensive animal protein sources. Therefore, if such an animal protein can be obtained from an insect, it is likely to have an excellent nutritional value to humans. Aspergillus oryzae and Bacillus licheniformis were fermentation starters for soy (or insect) meju. 12

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Additionally A. oryzae was used for defatted T. molitor larvae koji. Amino acids are mainly produced by proteases originating from these starters (Fu & Kim, 2011). This salt-tolerant protease was able to degrade insect protein resulting in release oligopeptides and free amino acids. Bacillus licheniformis strains were isolated and identified in meju used for soy sauce fermentation, and there were several studies in which this strain increased amino acids of soy sauce (Ham et al., 2004). Although it fermented for a relatively short-term, when the final product is industrially sterilized or

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treated with a preservative such as ethanol, it is possible to circulate the insect sauce at room temperature due to its high salt concentration as soy sauce.

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Preference test

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Color, flavor, taste, and overall acceptability of the sauces were measured on a 7-point scale (Table 7). No significant difference in color was observed among the samples. S8 and the commercial soy sauce (the positive control) had favorable flavors, but no significant difference was detected among

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samples. No significant difference in taste was detected among the samples, except S6. In the overall preference test, S6 had the lowest score. S8 and the commercial soy sauce had higher

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scores, but no significant difference was observed among samples. In addition, M6 had more lipids and roasted rice flour, and was the most preferable type among the insect sauces. The content of arginine of S6, a bitter tasting amino acid, was 225.36mg / 100g, which was more than 2 times higher

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than that of S8 and 10 times higher than that of insect soy sauce. Therefore, it seems to be low in

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sensory preference. Insect seasoning sauce is a liquid with no insect form of, and it contains amino acids and oligopeptides, expected to be more bioavailable, by decomposing insect protein through traditional microbial fermentation. It is anticipated that this type of product has a positive effect on

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4. Conclusion

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consumers to lower their rejection of insect food.

Insects are becoming a food for the future, and related studies carried out in various fields indicate that they are likely to be used as dietary protein sources. Currently insect protein production is not economically viable compared to the soybean industry, although it has a great deal of potential to the environment. This is mainly because the insect industry is still in the early stages of production, processing, and commercialization of the related products than other food industries. In order for insect proteins to be used as animal feed or edible food, economically viable mass production should be secured first, and technologies for processing and automation processes should be developed to 13

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make the insect industry economically competitive with the production of meat. The upscale soybean industry including soy sauce can be a model to test industrial-scale processing of insect protein (van Huis et al., 2013). In this study, a liquid fermented seasoning was prepared using T. molitor larvae by applying the soy sauce fermentation process. Meju, koji, and roasted rice flour were used with 23% brine to produce the sauce. A. oryzae and B. licheniformis fermented the raw or defatted insect larvae, and the resulting

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material was used as the insect meju. Insect koji was prepared with defatted insects and A. oryzae. Six sauce samples, including soy sauce with two different ratios of ingredients (meju:koji:roasted rice

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flour = 6:2:2 and 8:1:1) were prepared with raw T. molitor larvae (raw insect sauce), defatted larvae (defatted insect sauce), and soy (soy sauce) after 20 days of fermentation at 25°C. No difference was

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observed in the L value among the experimental groups. The 6:2:2 ratio sauces generally had higher L values than did the 8:1:1 ratio sauces. The raw insect sauce group had a high b value. No change in

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the ΔE value was observed over time, but this value was highest for the insect sauce. Browning tended to increase as fermentation continued in the soy and raw insect sauces. Browning of defatted insect sauce increased, but then decreased sharply on day 20. Sample pH values were 5.70–6.19 on

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day 0 and 5.66–6.02 on day 20. No significant pH change was observed among samples. Titratable acidity increased during fermentation. The 6:2:2 ratio sauces had more total soluble solids, reducing sugars, and total sugars than did the 8:1:1 ratio sauces. Total nitrogen content of the sauces after 20

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days of fermentation was 1.06–1.19%, exceeding the 0.70% total nitrogen value of the FAO/WHO

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Codex Alimentarius Commission’s international standard for soy sauce. The 6:2:2 ratio sauces had 0.26–0.32% amino-nitrogen content on day 20, which was greater than that in the 8:1:1 ratio sauces, indicating higher efficiency of protein degradation, but no significant difference from soy sauce.

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However, %NDR of the insect sauces was greater than that of the soy sauces. Essential and nonessential amino acids, as well as amino acid derivatives, increased 1.5–2 times during fermentation. M6 showed the highest total free amino acid value of 5104.17 mg/100g. The glutamic

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acid, alanine, aspartic acid, serine, isoleucine, lysine, phenylalanine, and valine contents were high in the sauces. Proline and glutamic acid were the most abundant amino acids, and citrulline content was much higher in the insect sauces than in the soy sauces. Preference evaluations did not differ between the insect and soy sauces. M6 was the most preferred among the insect sauces. Therefore, insect sauce similar to soy sauce is expected to be commercialized into a liquid seasoning form lacking the insect’s shape and texture characteristics, to reduce consumer rejection of the product.

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Acknowledgements This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through High Value-added Food Technology Development Project (316057-03-2-HD030), funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA), Korea. The authors thank for providing financial means to support this study

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Figure list Fig.1. Browning of soy and insect sauce 1)

Refer to sample legends for table 2.

Fig.2. Reducing sugar (%) of soy and insect sauce

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1) Refer to sample legends for table 2.

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Fig.3. Total sugar contents (%) of soy and insect sauce

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1) Refer to sample legends for table 2.

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4.5

3.5

PT

3.0

2.5

S8 S6 M8 M6 DM8 DM6

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2.0

1.0 0

5

10

15

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D

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Aging period(Day)

Fig.1.

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1.5

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Asorbance(420nm)

4.0

21

20

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1.4 S8 S6 M8 M6 DM8 DM6

1.0

0.8

PT

0.6

0.4

RI

0.2

0.0 6

13

20

SC

0

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Fermentation time(Day)

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D

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Fig.2.

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Reducing sugar(%)

1.2

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4.0 S8 S6 M8 M6 DM8 DM6

3.5

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2.5

2.0

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1.5

0.5 6

13

20

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Fermentation time(Day)

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D

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Fig.3.

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0

SC

1.0

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Total sugar (%)

3.0

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Table 1. Proximate analysis of soybean, and Tenebrio molitor larvae Crude fat

Moisture

Soybeans

9.20

40.47

11.26

34.82

4.25

(raw)

3.55

50.87

35.40

7.02

3.16

(defatted)

1.50

75.43

6.49

12.51

4.07

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CE

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D

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T. molitor larvae

24

Carbohydrate

Crude

Sample

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Crude protein

(Unit : %)

ash

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Table 2. Ingredients used to prepare the soy and insect sauces % weight of ingredients Sample1) Koji

Roasted rice flour

S8

22.9

2.9

2.9

S6

17.1

5.7

5.7

M8

22.9

2.9

2.9

M6

17.1

5.7

5.7

DM8

22.9

2.9

2.9

DM6

17.1

5.7

5.7

S8: soy sauce with soybean meju:koji:roasted rice flour = 8:1:1

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S6: soy sauce with soybean meju:koji:roasted rice flour = 6:2:2

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71.4

M8: raw insect sauce with mealworm meju0 koji:roasted rice flour = 8:1:1 M6: raw insect sauce with mealworm meju:koji:roasted rice flour = 6:2:2

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DM8: defatted insect sauce with defatted mealworm meju:koji:roasted rice flour = 8:1:1

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D

DM6: defatted insect sauce with defatted mealworm meju:koji roasted rice flour = 6:2:2

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1)

23% brine

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Meju

25

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Table 3. Color parameters of the soy and insect sauces Da

S83)

46.76 ± 0.26b2)

45.76 ± 2.05ca

46.76 ± 1.45a

44.91 ± 0.65

S6

47.63 ± 1.12b

44.58 ± 0.38a

45.51 ± 0.36a

44.76 ± 0.08

M8

43.17 ±

0.55c

1.33ab

0.77a

46.19 ± 1.49

M6

49.76 ± 0.16a

50.83 ± 0.31a

47.03 ± 0.56ab

46.71 ± 3.84

DM8

43.50 ± 0.05c

48.47 ± 0.63ab

43.06 ± 0.76bc

44.33 ± 0.33

DM6

44.20 ± 0.10c

47.21 ± 0.31bc

44.22 ± 1.22c

F-value

99.80***1)

17.76***

13.57***

S8

1.51 ± 0.09c

1.97 ± 0.14c

2.30 ± 0.21d

2.43 ± 0.12c

S6

1.79 ± 0.44bc

2.44 ± 0.08bc

2.79 ± 0.28c

2.63 ± 0.02bc

M8

2.10 ± 0.13b

2.70 ± 0.27ab

2.84 ± 0.11c

3.43 ± 0.63ab

M6

1.99 ± 0.01b

3.03 ± 0.08a

3.50 ± 0.14b

3.88 ± 0.64a

DM8

3.79 ± 0.02a

2.02 ± 0.51c

DM6

3.78 ± 0.05a

2.86 ± 0.08ab

F-value

113.77***

12.47***

S8

11.03 ± 0.57a

S6

45.39 ± 0.95

SC

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47.33 ±

NS

3.98 ± 0.05a

3.93 ± 0.22a

4.19 ± 0.08a

50.94***

15.77***

7.10 ± 0.55bc

4.59 ± 1.20c

6.23 ± 0.45b

9.78 ± 1.48ab

8.27 ± 0.64ab

7.70 ± 2.61b

6.32 ± 0.05b

M8

7.76 ± 0.25cd

9.83 ± 1.22a

9.62 ± 0.53ab

11.45 ± 1.49a

M6

6.57 ± 0.06d

10.59 ± 0.65a

11.29 ± 2.77a

DM8

9.55 ± 0.08ab

3.34 ± 1.19d

7.34 ± 0.35bc

7.71 ± 0.26b

DM6

9.00 ± 0.00bc

5.49 ± 0.17c

6.94 ± 0.63bc

8.18 ± 0.64b

F-value

23.09***

36.47***

11.32***

12.30***

S8

48.07 ± 0.13b

46.35 ± 2.11cd

47.05 ± 1.32ab

45.40 ± 5.77ab

48.68 ± 0.83b

45.41 ± 0.49d

46.29 ± 0.78bc

45.28 ± 0.75ab

43.91 ± 0.59d

49.65 ± 1.05ab

48.39 ± 0.65a

47.74 ± 1.04ab

AC

M8

MA

3.96 ± 0.12a

S6

ΔE

48.58 ±

PT

20

D

b

13

9.07 ± 0.49a

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a

6

CE

L

0

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y sample

M6

50.23 ± 0.16a

51.72 ± 0.21a

48.33 ± 0.40a

48.32 ± 3.02a

DM8

44.70 ± 0.03cd

48.63 ± 0.71bc

43.86 ± 0.69d

45.17 ± 0.32b

DM6

45.27 ± 0.10c

47.62 ± 0.30bcd

44.93 ± 1.11cd

46.31 ± 0.82ab

F-value

140.62***

19.64***

17.25***

3.91*

1) NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 2) a–d means in a column with different superscripts differ significantly at the 5% level (Tukey’s HSD test). 3) Refer to the sample codes in Table 1.

26

ACCEPTED MANUSCRIPT

Table 4. pH, triable acidity, salinity, and total soluble solids of soy sauce and insect sauce

Fermentation time (days)

Sample

0

S63)

0.00b

5.95 ±

0.04a

5.95

5.53 ± 0.01c

5.56 ± 0.01c

5.66 ± 0.02

M8

5.70 ± 0.03d

5.72 ± 0.02b

5.70 ± 0.03b

5.73 ± 0.01

5.70 ±

0.00d

5.66 ±

0.05b

0.00bc

5.70 ± 0.00

5.75 ±

0.00c

5.69 ±

0.03b

0.00b

5.84 ± 0.15

5.65 ±

58.152***

RI

90.103***

5.65 ±

6.02

PT

6.10 ±

654.400***1)

6.08 ±

5.97 ±

5.74 ± 0.00cd

F-value

NS

0.07e

0.55 ± 0.06ab

0.47 ± 0.04d

0.51 ± 0.00b

S6

0.05

0.14 ± 0.02

S8

0.03

0.11 ± 0.03

M6

0.10

0.18 ± 0.04

0.56 ± 0.04cd

0.61 ± 0.02a

M8

0.10

0.17 ± 0.04

0.62 ± 0.04bc

0.61 ± 0.02a

0.00ab

0.32 ±

0.60 ± 0.00a

DM6

0.10

0.18 ± 0.04

0.68 ±

DM8

0.10

0.19 ± 0.05

NU

0.77 ± 0.04a

0.59 ± 0.02a

NS

51.824***

6.883**

18.90 ± 1.55ab

F-value S6

20.74 ± 0.04d

S8

20.92 ±

0.12cd

M6

21.32 ±

0.04ab

M8

20.36 ± 0.00

17.29 ±

21.88 ± 1.02

21.06

21.12 ±

0.24a

21.73 ± 0.78

20.77 ± 0.15

21.06 ± 0.00bc

20.83 ± 0.96a

22.51 ± 0.96

20.68 ± 0.11

DM6

21.27 ± 0.04ab

20.24 ± 0.91a

22.23 ± 0.13

21.03 ± 0.53

DM8

0.13a

0.18a

D

MA

21.88 ± 1.13

1.53b

22.63 ± 0.12

20.80 ± 0.20

8.023***

NS

NS

37.50 ± 4.12

45.00 ± 3.46

42.00 ± 0.00a

35.00 ± 0.00b

35.00 ± 3.46

45.50 ± 3.00

40.00 ± 0.00a

36.00 ± 0.00ab

37.00 ± 1.15

41.00 ± 1.15

37.00 ± 1.15b

36.50 ± 0.71a

37.00 ± 1.15

43.00 ± 2.58

36.50 ± 1.00b

DM6

35.00 ± 0.00b

37.50 ± 1.91

42.00 ± 0.00

40.00 ± 0.00a

DM8

0.00b

36.50 ± 1.00

42.00 ± 0.00

37.50 ± 1.00b

NS

NS

18.704***

S6 S8 M6

CE

M8

21.44 ±

23.891**

PT E

F-value

Total soluble solids (°Brix)

0.02a

20

S8

DM8

Salinity (%)

6.00 ±

0.05a

M6

DM6

Titratable acidity (%)

6.19 ±

13

0.08a2)

SC

pH

6 0.00a

AC

F-value

35.00 ±

35.000 ±

00b

20.74 ±

10.600**

1) NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 2) a–c Means in a column with superscripts differ significantly at the 5% level (Tukey’s HSD test). 3) Refer to sample codes in Table 1.

27

ACCEPTED MANUSCRIPT

Table 5. Changes in total nitrogen, amino nitrogen, and NDR 1) in soy sauce and insect sauce during fermentation. Day sample S84)

0.71 ± 0.01c

1.20 ± 0.02ab

1.14 ± 0.01bc

1.20 ± 0.03b

S6

0.68 ± 0.00c

1.16 ± 0.01b

1.11 ± 0.00cd

1.31 ± 0.02a

M8

0.73 ± 0.00c

1.26 ± 0.01a

1.21 ± 0.00ab

1.06 ± 0.01c

M6

0.73 ± 0.00c

1.14 ± 0.05b

1.04 ± 0.01d

1.06 ± 0.01c

DM8

0.98 ± 0.03a

1.25 ± 0.00a

1.28 ± 0.02a

DM6

0.89 ± 0.00b

1.13 ± 0.01b

1.16 ± 0.04bc

F-value

114.50***2)

16.81*

35.38***

23.12**

S8

0.05 ± 0.00

0.25 ± 0.03c

0.27 ± 0.10

0.27 ± 0.00cd

S6

0.05 ± 0.00

0.27 ± 0.04bc

M8

0.04 ± 0.00

0.34 ± 0.03ab

M6

0.05 ± 0.00

0.28 ± 0.01abc

DM8

0.06 ± 0.01

0.35 ± 0.02a

DM6

0.09 ± 0.04

0.33 ± 0.05ab

F-value

NS

S8

PT

20

1.19 ± 0.03b

SC

RI

1.19 ± 0.04b

0.32 ± 0.01ab

0.23 ± 0.02

0.26 ± 0.01d

0.21 ± 0.00

0.29 ± 0.02bc

0.28 ± 0.04

0.32 ± 0.01ab

0.25 ± 0.01

0.32 ± 0.00a

6.64**

NS

23.78***

7.09 ± 0.52

18.98 ± 1.65b

17.23 ± 2.45b

22.47 ± 0.56d

S6

7.88 ± 0.06

24.94 ± 0.82a

19.62 ± 0.42ab

24.13 ± 0.36cd

M8

5.51 ± 0.04

25.53 ± 1.88a

20.08 ± 0.14ab

25.69 ± 0.13bc

M6

7.35 ± 0.05

24.69 ± 1.07a

19.79 ± 0.08ab

28.59 ± 0.38a

DM8

5.79 ± 1.25

27.81 ± 0.86a

22.15 ± 0.96a

26.75 ± 0.38ab

DM6

10.61 ± 4.31

26.67 ± 0.49a

21.40 ± 0.09ab

27.21 ± 1.03ab

F-value

NS

15.58*

4.86*

32.78***

MA

NU

0.25 ± 0.03

CE

NDR

13

D

AN

6

PT E

TN

0

1) NDR, nitrogen degradation ratio

AC

2) NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 3) a–d Means in a column with different superscripts differ significantly at the 5% level (Tukey’s HSD test). 4) Refer to sample codes in Table 1.

28

ACCEPTED MANUSCRIPT

Table 6. Free amino acid contents of soy and insect sauces prepared at different ratios Amino acid

S6

S8

M6

M8

(mg/100g)

DM6

DM8

Arginine

225.36

100.84

19.84

9.58

79.31

32.30

Histidine

166.12

138.09

193.06

172.58

161.53

171.95

Isoleucine

268.41

198.62

298.04

271.97

246.44

260.84

Leucine

350.67

267.09

404.43

376.23

349.98

360.22

Essential

Lysine

265.04

240.45

280.40

259.38

248.19

255.99

amino acids

Methionine

98.29

65.82

113.90

81.86

68.74

72.93

Phenylalanine

292.00

218.81

270.35

206.52

Threonine

230.44

144.96

292.01 114.70

123.16

64.89

73.22

49.89 195.67

391.81

354.98

314.17

340.65

2246.35

1620.25

2378.54

2102.18

1954.50

2020.28

Alanine

218.80

153.03

362.46

326.81

305.66

321.45

Aspartic acid

379.50

249.95

337.39

246.40

234.15

Asparagine

12.17

4.41

17.12

23.35

8.78

14.00

Glutamic acid

698.95

574.96

653.36

Glycine

164.46 86.79

Serine

269.17

Tyrosine

112.90

Cysteine

61.25

SC

247.88 514.12

504.26

531.78

119.56

185.20

149.95

171.88

178.19

63.34

591.48

645.20

401.16

563.89

168.01

311.10

246.20

230.23

238.82

93.04

213.06

125.82

129.68

155.95

32.96

54.47

30.12

38.54

36.10

2003.98

1459.25

2725.63

2309.43

2036.58

2274.32

Hydroxyproline

70.72

45.00

58.17

46.82

46.36

42.33

Hydroxylysine

39.97

13.78

16.16

18.47

19.55

28.38

31.54

17.58

14.86

20.06

15.44

D

Total

MA

Proline

RI

60.45 289.59

NU

PT

247.39

Valine

amino acids

32.30

14.06

2.22

11.87

4.48

8.83

3.18

β-aminoisobutyric acid

31.35

9.90

84.04

70.12

42.95

44.44

γ-amino-n-butyric acid

39.17

48.63

5.85

6.18

6.33

8.92

1-methylhistidine

1.54

1.74

0.88

0.36

1.00

0.96

3-methylhistidine

4.92

3.34

6.05

4.44

4.75

4.95

Carnosine

4.66

3.34

2.37

1.41

3.36

2.54

Citrulline

359.64

266.88

423.97

290.53

288.58

296.67

Taurine

1.29

3.32

6.73

6.80

4.96

6.94

Ornithine

70.09

79.48

123.40

129.39

94.47

117.04

Phosphoserine

27.98

22.34

34.02

28.94

25.96

29.15

α-aminoadipic acid

AC

CE

PT E

α-aminobutyric acid

Derivatives

204.79

228.19

Tryptophan Total

Nonessential

193.07

245.93

Phosphoethanolamine

7.32

6.97

0.22

0.37

0.79

0.43

Sarcosine

50.68

38.85

29.72

30.37

27.72

21.67

Urea

165.60

147.79

125.63

41.84

72.77

50.01

20.75

20.02

68.40

75.09

48.45

65.88

Total

Ethanolamine

938.26

763.77

1012.91

768.44

716.10

730.45

Total amino acid

5188.59

3843.27

6117.08

5180.05

4707.18

5025.05

29

ACCEPTED MANUSCRIPT

Table 7. Consumer preference for the soy and insect sauces sample Color

Flavor

Taste

Overall acceptance

S83)

4.79 ± 1.48

4.36 ± 1.34a 2)

4.71 ± 1.33a

4.43 ± 1.34a

S6

3.79 ± 1.37

2.93 ± 1.82b

2.21 ± 1.42b

2.50 ± 1.74b

M8

4.30 ± 1.62

3.95 ± 1.50ab

4.25 ± 1.69a

3.91 ± 1.60a

M6

3.95 ± 1.80

4.05 ± 1.40ab

4.43 ± 1.42a

3.95 ± 1.43a

DM8

3.82 ± 1.74

3.22 ± 1.42ab

3.55 ± 1.47ab

DM6

4.11 ± 1.74

3.66 ± 1.71ab

3.68 ± 1.65a

CS

4.40 ± 2.03

4.40 ± 1.92a

4.40 ± 1.63a

F-value

NS1)

2.423*

PT

attributes

3.51 ± 1.48ab

SC

RI

3.74 ± 1.69ab 4.60 ± 1.35a

5.437***

1) NS: not significant, *P < 0.05, **P < 0.01, ***P < 0.001

NU

2) a–c Means in a column with different superscripts differ significantly at the 5% level (Tukey’s HSD test).

AC

CE

PT E

D

MA

3) Refer to sample codes in Table 1. CS: commercial Korean brand soy sauce.

30

3.082**

ACCEPTED MANUSCRIPT

Highlights -

New seasoning sauce was developed using mealworm (Tenebrio molitor) larvae by applying the soy sauce fermentation process.

-

Characteristics of the sauce during fermentation were monitored and compared to soy sauce.

PT

The percentage nitrogen degradation rates were higher for the insect protein than for the

CE

PT E

D

MA

NU

SC

RI

soy protein.

AC

-

31