**Antiradical Activity of Hydrolysates and Extracts from Mollusk** *A. broughtonii* **and Practical Application to the Stabilization of Lipids**

### **O.V. Tabakaeva 1,\*, W. Piekoszewski 1,2,\*, T.K. Kalenik 1, S.N. Maximova 3, A.V. Tabakaev 1, D. V. Poleshyk <sup>3</sup> and L. Proniewicz <sup>4</sup>**


Received: 29 December 2019; Accepted: 29 February 2020; Published: 7 March 2020

**Abstract:** The antiradical properties of hydrolysates and hydrothermal extracts of bivalve mollusks (*Anadara broughtonii*) from the Far Eastern Region of Russia and their influence on lipid oxidation in mayonnaise were investigated. The radical binding activity of hydrolysates and extracts of *A. broughtonii* varies from 55% to 89%. The maximum radical-binding activity was observed for acid hydrolysates. The antiradical efficiency of acid hydrolysates is 35%–41% of the BHT (butylhydroxytoluene) index. The antiradical activity depends on the (method of) technological and biotechnological processing of raw materials. Acid and enzymatic hydrolysates and hydrothermal extracts of *A. broughtonii* in mayonnaise slow down the process of oxidation of lipids and hydrolysis of triglycerides. Acid hydrolysates reduce the speed of oxidation and hydrolysis of lipids in mayonnaise more efficiently than the enzymatic hydrolysates.

**Keywords:** antiradical activity; hydrolysates; hydrothermal extracts; *A. broughtonii*

#### **1. Introduction**

The study of free-radical oxidation processes is important today due to their proven influence on various metabolic processes of the human body [1–3]. The accumulation of free radicals causes lipid peroxidation and dysfunction of the cell membrane, which leads to premature aging of the body, frequent sickness and even malignant tumors [4–6]. A quantitative study of the antiradical properties of given substances can suggest the ways in which such substances with antiradical activity are applied in practice. However, up till now, the focus of studies on antiradical activity has been mainly oriented towards vegetable raw materials—essential oils and mixtures thereof, and extracts of medicinal and other plants [7–9]. At the same time, raw materials of animal—including marine—origin, and products (of animal/marine origin) that have been subjected to technological and biotechnological modification could also have antioxidants. The biological and pharmacological activities of organic natural compounds of marine origin have been demonstrated by numerous studies [10–13].

Nowadays, many species of marine organisms that are used for food purposes, in traditional and non-traditional medicine, were also shown to have antioxidant activity and as raw materials for obtaining biologically active additives. Amongst marine hydrobionts, bivalve mollusks are promising sources of biologically active substances with antiradical properties. Substances from bivalves exhibit high antioxidant capacity: Extracts from *Crassula aequilatera*, *Mactra murchisoni*, and *Paphies donacina*, which were reported to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals [14]; *Paphia malabarica* extracts, which were evaluated in relation to DPPH and ABTS<sup>+</sup> radical [15] crude extracts and fractions of alcohol extracts from *Mactra veneriformis* [16]; and oligopeptides from *Meretrix meretrix* [17].

The clams *Anadara broughtonii*, *Spisula sachalinensis*, *Mactra chinensis*, and others can be found in the coastal zone of the Sea of Japan [18]. The tissues of bivalve mollusks are rich in amino acids and specific carbohydrates [19–25]. *A. broughtonii* belongs to the family *Arcidae* and is a rather common type of Mollusca, in the *Bivalvia* class. It is found in the upper-sublittoral in subtropical areas of the Asian Pacific, with commercial stocks at depths of 2–15 m. The maximum lifespan of *A. broughtonii* is 65 years; the total mass of individuals varies from 80 to 380 g, and the shell length is 65–80 mm. *A. broughtonii* inhabits muddy and sandy bottoms, burrowing to depths of 10–25 cm. It is found in the Yellow Sea and the Sea of Japan [26].

Acid and enzymatic hydrolysates and hydrothermal extracts are products of technological and biotechnological processing of bivalve mollusks. They are sources of biologically active substances with potential health benefits [27,28].

Their antioxidant properties may be the basis for use as ingredients in lipid-containing emulsion products in order to reduce the rate of oxidation of lipids. Nowadays, various synthetic substances that perform the function of antioxidants are used for lipid stabilization of vegetable oils—potassium diethyl dithiophosphate or 2,4,6-tris-(dimethylamino) phenol or antioxidant mixtures containing BHT, naphthols, propyl gallates, and others. The disadvantages of these stabilizers are that they are not devoid of toxic properties [29]. Natural antioxidants are safer at stabilizing the quality of lipid rich foods [30].

The purpose of the present study was to investigate the antiradical properties of acid and enzymatic hydrolysates and hydrothermal extracts of the soft tissues of the bivalve mollusk *A. broughtonii* and to determine their effect on the oxidation of lipids in mayonnaise.

#### **2. Materials and Methods**

#### *2.1. Material from the Bivalve Mollusk A. broughtonii*

The clam *A. broughtonii* ranges from 65 to 80 mm in length, and its mass varies from 80 to 200 g. Molluscs were collected in June, September, and November 2016, and in February and April 2017 from Amur Bay (43060 N and 131440 E), Sea of Japan, in the Primorsky region in Russia (all analyses were done using pooled mollusks—seasonal changes were not taken into consideration in this study). The mollusks from all the sampling months were pooled to one sample of 5 kg. Live bivalve mollusks were transported under refrigeration (+6 ◦C) to the laboratory within 3 h and sampled randomly for this study. Upon arrival, the clams were manually shucked by cutting the adductor muscle with a knife. The clam juice was removed and the edible portion, constituting 12.46%–14.08% of the total weight of *A. broughtonii*, was collected. The edible portion was then dissected into 4 parts: Muscle, mantle, adductor, and viscera. Muscle and mantle were powdered using a blender (Phillips, Guangzhou, China) in the presence of liquid nitrogen. The samples were packed in a polyethylene bag, sealed and stored at −20 ◦C until use. The storage time was no longer than 1 month.

#### *2.2. Acid and Enzymatic Hydrolysis and Hydrothermal Extraction and Analysis*

Hydrolysates and extracts were obtained separately from the mantle and muscle of the mollusk. The frozen tissues of bivalve mollusks *A. broughtonii* were thawed at 4 ◦C before use, mixed with 6% solution of citric acid at a proportion of 1:1 according to the mass of tissues and acid, then homogenized in a tissue triturator for 2 min, and homogenate was obtained. The hydrolysis reaction was performed at 95 ◦C for 8 h. The resulting liquid phase was an acid hydrolysate (AH). In order to obtain enzymatic hydrolysate (EH), tissues of *A. broughtonii* were mixed with deionized water, the proportion being 1:2 according to the mass of tissues and water, then homogenized in a tissue triturator for 2 min, and

homogenate was obtained. A hydrolysis reaction was performed at 45 ◦C and pH 7.5 in a shaking incubator at 150 rpm in order to promote activity of enzymes. The proteolytic enzyme Protomegaterin G20x (TU 00479942-002-94, Russia), produced by *Bacillus megaterium* was obtained from OOO PO SibBioPharm (Berdsk, Russia). Protomegaterin is a food-grade enzyme which has a declared proteolytic activity of 800 U/g. The enzyme was stored at 4 ◦C until it was used for the hydrolysis experiments.

The hydrolysis time was 8 h and the enzyme:substrate ratio was 5:1000. At the end of the hydrolysis, the liquid phase was separated by filtration, and boiled for 20 min in order to inactivate the enzyme. In order to obtain hydrothermal extracts (HTE), *A. broughtonii* tissues were mixed with deionized water in a 1:1 ratio according to the mass of tissues and water, and were then homogenized in a tissue triturator for 2 min, and homogenate was obtained. High-temperature processing was performed at 100 ◦C, duration 3 h. The moisture content was measured according to methodology described by the Association of Official Analytical Chemists (AOAC) (2000). Samples were dried in an oven at +105 ◦C until a constant weight was obtained [31].

#### *2.3. Preparation of Mayonnaise*

The mayonnaise ingredients included vegetable oil, egg powder, acid or enzymatic hydrolysate or hydrothermal extract, citric acid, mustard powder, salt, sugar, and water. The concentrations of additives (acid, enzymatic hydrolysates, hydrothermal extract) were determined on the basis of organoleptic indicators, primarily taste. The mayonnaise recipe is presented in Table 1.


**Table 1.** Composition of mayonnaise with hydrolysates and hydrothermal extracts from mollusks (*A. broughtonii*).

Loose components (salt, sugar, citric acid, dry egg powder) were sieved and weighed out in accordance with recipes, then poured into food containers. Citric acid, sugar, and sodium chloride were dissolved in the required amount of water. In the case of using acid hydrolysates in the recipe, the procedure for the preparation of citric acid solution was excluded from the scheme of the technological process. The egg powder was mixed with water at a temperature of 40–45 ◦C and a ratio of 1:2, was heated to 60–65 ◦C, was held for 20–25 min for pasteurization, and then cooled to 30–40 ◦C. The acid hydrolysates or enzymatic hydrolysates or hydrothermal extracts, at a temperature of 55–60 ◦C, were mixed with the egg powder, mustard powder, and the sugar grains. The compound was stirred thoroughly and heated to 55–60 ◦C for 25–30 min in order to prepare the mayonnaise paste. After obtaining the mayonnaise paste and cooling it, an emulsion of mayonnaise was prepared. Vegetable oil at a temperature of 20 ◦C was injected into the mayonnaise paste while stirring. An acid-salt or brine solution was added after adding the whole oil dose, and the mixture was stirred for 15–20 min in order to further homogenize the emulsion. Final homogenization of the resulting mixture was carried out using a homogenizer (Kinematica, Polytron PT 45–80 GT, Switzerland). The pH value of mayonnaise

was: 3.9 with acid hydrolysates, 4.1 with enzymatic hydrolysates, 4.3 with hydrothermal extracts, 3.9 with BHT, and 4.2 without additives. The samples were kept at room temperature (22 ± 2 ◦C). At designated times (30, 50, 70 and 90 days), the samples were taken for analyses. Mayonnaise without acid hydrolysates or enzymatic hydrolysates or hydrothermal extracts was used as a control. As a positive control, mayonnaise with the addition of BHT (Aldrich, 99.9%, analytical grade) was used.

#### *2.4. Fractionation of Melanoidins*

For fractionation of melanoidins, we used the method of gel-chromatography on columns with TSK-gels Toyopearl HW-40 and HW-50 ("Toyo Soda", Japan), pre-calibrated for proteins with well-known molecular weights. The gels that were used had the following ranges for separation of molecular masses for proteins: The separation of proteins with molecular masses from 100 to 10,000 Da takes place on HW-40; and on HW-50 for proteins from 500 to 80,000 Da. A 0.2 M solution of sodium chloride and distilled water was used as eluent. Samples hydrolysates and hydrothermal extracts were applied on the column (1.2 × 35 cm, free volume 12 mL) in 0.4 mL pre-skim chloroform.

The optical density of fractions of volume 1 mL was measured using a scanning spectrophotometer UV-1800 (Shimadzu, Japan) at 400 and 420 nm wavelengths. Calculation of the melanoidins content was carried out according to formulas derived on the basis of the standard curve [32].

#### *2.5. DPPH Radical Scavenging Assay*

Antiradical properties were evaluated in terms of ability to interact with the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro. Determination was carried out in a reaction mixture containing 3 mL of 0.3 mM DPPH in ethanol, 1 mL 50 mM tris -HCl- buffer, pH 7.4, and 1 mL of extract or hydrolysate [33]. After 30 min incubation at a temperature of 20 ◦C, values of optical density were recorded at λ = 517 nm. The experiments were performed on a scanning spectrophotometer UV 1800 (Shimadzu, Japan) in cuvettes (l = 1 cm) at T = 298 ◦K

The activity was characterized by the following indicators:


$$\text{RBA } \text{(\%)}=\text{(D}\_{517}\text{I}-\text{D}\_{517}\text{II)/D}\_{517}\text{I}\times100,\tag{1}$$

where D517I is a control, and D517II is a sample;


$$\text{AE} = 1/(\text{E}\_{\text{C50}} \times \text{T}\_{\text{EC50}}) \tag{2}$$

Antiradical properties were compared with the effect of the well-known synthetic antioxidant BHT (2,6-ditretbutyl-4-methyl-phenol), which was previously purified by recrystallization from ethanol, and then the isolated crystals were dried and sublimed in a vacuum.

#### *2.6. Acid and Peroxide Value (AV, PV)*

The acid and peroxide values (AV, PV) in the oil samples, in the oil samples extracted from mayonnaise with a mixture of solvents (ethyl ether and chloroform) were determined according to the methods stipulated in ISO [34,35]. The peroxide values were measured spectrophotometrically at 500 nm by a UV–1800 instrument (Shimadzu, Japan). Results were expressed in mmol of oxygen per kilogram of oil. Acid value (AV) is an important indicator of vegetable oil quality. AV is expressed as the amount of KOH (in milligrams) necessary to neutralize free fatty acids contained in 1 g of oil.

#### *2.7. Statistical Analysis*

Results were expressed as mean values (standard deviation) (n=9). In total, three acid hydrolysates, three enzymatic hydrolysates, and three hydrothermal extracts were obtained. Data were subjected to analysis of variance (ANOVA), and mean comparisons were carried out using the Duncan's multiple range test (hydrolysates and hydrothermal extracts) and t-test (samples of mayonnaise with or without additives). Statistical analysis was performed using the statistical Package for Social Sciences (SPSS for windows: SPSS Inc., Chicago, IL, USA). Results with *p* < 0.05 were considered to be statistically significant.

#### **3. Results and Discussion**

The acid and enzymatic hydrolysates and hydrothermal extracts of motor muscle and mantle of *A. broughtonii* represent a dark brown liquid, with a specific faint odor. The content of dry substances was 15.9%–16.3% in acid hydrolysates, 8.3%–8.9% in enzymatic hydrolysates, and 5.6%–5.9% in hydrothermal extracts. The appearance of the received hydrolysates and extracts is shown in Figure 1.

**Figure 1.** Appearance of hydrolysates and extracts (**A**—acid hydrolysate (AH) muscle, **B**— enzymatic hydrolysates (EH) mantle, **C**—hydrothermal extracts (HTE) muscle).

Evaluation of the antiradical activity of the obtained acid and enzymatic hydrolysates and hydrothermal extracts of motor muscle and mantle of *A. broughtonii* and the well-known antioxidant BHT showed that all the studied objects have a high ability to bind radical DPPH (Table 2).

The radical-binding activity of acid and enzymatic hydrolysates and hydrothermal extracts of motor muscle and mantle of *A. broughtonii* varied within wide limits: From 55% to 89%. The results clearly indicate that the acid hydrolysate of mantle exhibited the highest radical binding activity (89%); this value is only 5% lower than for BHT. The minimum antiradical activity was shown by hydrothermal extracts of muscle, and this is characteristic for the extracts obtained from different parts of the bivalve mollusk *A. broughtonii*. Enzymatic hydrolysates possess lower antiradical activity than acid hydrolysates, but slightly higher than hydrothermal extracts. Our study showed that the method of modifying tissue of the bivalve mollusk *A. broughtonii* more significantly influences the level of manifestation of antiradical properties than the biological raw materials used. The data obtained are presented in Table 2.


Antiradicalactivityofhydrolysatesandextractsfrommollusk*A.*

#### *Foods* **2020** , *9*, 304

The data in Table 2 demonstrate that acid hydrolysates have high antiradical efficiency (AE), and, furthermore, that acid hydrolysates from the mantle have higher values than those from muscle. The antiradical efficiency of acid hydrolysates is 35%–41% of the AE of BHT, which is a good indicator of antiradical properties. The acid hydrolysates of the mantle have 89% more AE than enzymatic hydrolysates, and 179% more than hydrothermal extracts. The AE of acid hydrolysates of muscle is 29% greater than that of enzymatic hydrolysates of muscle and 2.05 times greater than that of hydrothermal extracts (of muscle).

Similarly to our result, the protein hydrolysate obtained from the oyster *Saccostrea cucullata* exhibited 85.7% [10], the hydrolysate of the common smooth hound (*Mustelus mustelus*) exhibited 76.7% of scavenging activity [36], and protein hydrolysate of *Conus betulinus* exhibited 84% of scavenging activity [37]. By comparing the percentage of radical scavenging activity, the acid and enzymatic hydrolysates have proven to be a potential source for antiradical activity.

It is known that melanoidins and free amino acids exhibit antiradical properties. Previous research [38] has shown that acid hydrolysates of the tissues of mussels contain free amino acids, amines, dipeptides, free fatty acids, mineral substances, and high molecular substances, which are represented by two fractions: I—a molecular weight of 1500–6000 Da, and II—a weight less than 1500 Da. Moreover, the biological activity of fraction II was expressed significantly more strongly than fraction I. It was shown that fraction II comprises melanoidins having biological activity.

The fractional composition of high-molecular substances—acid and enzymatic hydrolysates and hydrothermal extracts—was researched in the present study. The quantitative proportions of fractions of melanoidins are presented in Figure 2.

**Figure 2.** The proportion of melanoidins fractions in hydrolysates and extracts of bivalve mollusk *A. broughtonii* (1—AH muscle, 2—AH mantle, 3—enzymatic hydrolysate (EH) muscle, 4—EH mantle, 5—HTE muscle, 6—HTE mantle). Data are mean ± standard deviation (n = 9). letters indicate which fraction has the highest content of dry solids. a—fraction I versus fraction II (*p* < 0.05). b—fraction I versus fraction III (*p* < 0.05). c—fraction II versus fraction III (*p* < 0.05).

#### *Foods* **2020**, *9*, 304

High molecular substances are divided into three fractions—two of them are substances with a molecular mass of about 10,000 Da, and the third fraction is of molecular weight of about 1000 Da. The maximum content of melanoidins was found in fraction III; fractions I and II contained only a small amount of melanoidins. The acid extract from the mantle was characterized by the highest content of melanoidins in fraction III.

Synthetic antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate are often added to foods to slow lipid oxidation.

However, concerns have been raised about their safety as additives in food. Therefore, natural antioxidants with little or no side effects are of some interest as health-friendly supplements that can preserve food quality, primarily to protect fats from oxidation [39–41].

It is known that protein hydrolysates and peptides may exhibit antioxidant activity against lipid and fat peroxidation [39]. It has also been shown that hydrolysates and peptides of proteins of marine origin exhibit antioxidant activity in vitro and even have higher antioxidant activity than α-tocopherol [42–44].

Therefore, hydrolysates and hydrothermal extracts of bivalves can be used as a functional ingredient in the food industry, in particular in the production of mayonnaise.

Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and ethylenediaminetetraacetic acid (EDTA) (commercial antioxidants) are widely used in mayonnaise to prevent spoilage and the appearance of a bitter taste. However, these additives have a carcinogenic effect when used in high concentrations [45]. Consumers are demonstrating a growing demand for mayonnaise with natural ingredients. Adding natural antioxidants to food oil-fat emulsion systems, such as mayonnaise, has great potential to increase their oxidative stability and will be attractive to a wider group of consumers. Also, these compounds can have an impact on health, which will position mayonnaise as a healthy and natural product [46]. Gallic acid [47], ascorbic acid [48], tocopherol [49], rosemary [50], lactoferrin [51,52], phitic acid [52], mustard [53,54] lycopene [55], ginger powder [56], grape seed extract [57], essential oils extracted from Zenyan [58], chitosan [59], and seaweed [46] have been evaluated as natural antioxidants in mayonnaise.

However, protein hydrolysates are not used as antioxidants in the composition of mayonnaise. In the present study, acidic, enzymatic hydrolysates, and hydrothermal extracts in the composition of mayonnaise significantly inhibited lipid oxidation processes, as evidenced by a decrease in peroxide value compared to the control for all studied samples, but the value of the positive control was lower (Figure 3).

The minimum peroxide value (PV) during the storage period is determined for the fat released from the mayonnaise with acidic hydrolysates, which correlates well with the high antiradical properties of the hydrolysate established earlier. The enzymatic hydrolysates and hydrothermal extracts in mayonnaise also reduce the PV of mayonnaise lipids in proportion to their antiradical properties described above. Acid and enzymatic hydrolysates and hydrothermal extracts in mayonnaise reduce PV (90 days of storage) by 27%–50%. According to the data [54], lycopene crystals containing 50 mg/kg in mayonnaise reduced the peroxide number of mayonnaise by 54% during the three-month storage period. However, a ginger powder mass fraction in mayonnaise of 1% reduced the peroxide number by only 25% during 4 months of storage [55]. Thus, acid hydrolysates reduced the peroxide number as lycopene, hydrothermal extracts as ginger powder. Mayonnaise without additives had a shelf life of no more than 90 days; the peroxide number at the end of the shelf life exceeded 20 mmol O2/kg. The shelf life of such mayonnaise was 17 days less than mayonnaise with additives of hydrolysates and hydrothermal extracts from the bivalve mollusk *A. broughtonii*.

The dynamics of changes of the acid value of fat isolated from mayonnaise with additives (acid, enzymatic hydrolysates and hydrothermal extracts and BHT) and without additives, during storage is presented in Figure 4.

**Figure 3.** The peroxide value depending on the storage duration 1—mayonnaise with AH muscle, 2—mayonnaise with EH muscle, 3—mayonnaise with HTE muscle, 4—mayonnaise without additives (negative control), 5—mayonnaise with butylated hydroxytoluene (BHT) (positive control), 6—mayonnaise with AH mantle, 7—mayonnaise with EH mantle, 8—mayonnaise with HTE mantle). Data are mean ± standard deviation (n = 9). a—mayonnaise with AH muscle versus mayonnaise without additives (negative control); b—mayonnaise with AH mantle versus mayonnaise without additives (negative control); c—mayonnaise with EH mantle versus mayonnaise without additives (negative control); d—mayonnaise with EH muscle versus mayonnaise without additives (negative control), e—mayonnaise with HTE muscle versus mayonnaise without additives (negative control), f—mayonnaise with HTE mantle versus mayonnaise without additives (negative control), and g—mayonnaise with BHT (positive control) versus mayonnaise without additives (negative control) (*p* < 0.05).

**Figure 4.** The acid value depending on the storage duration (1—mayonnaise with AH muscle, 2—mayonnaise with EH muscle, 3—mayonnaise with HTE muscle, 4—mayonnaise without additives (negative control), 5—mayonnaise with BHT (positive control), 6—mayonnaise with AH mantle, 7—mayonnaise with EH mantle, 8—mayonnaise with HTE mantle). Data are mean ± standard deviation (n = 9). a—mayonnaise with AH muscle versus mayonnaise without additives (negative control); b—mayonnaise with AH mantle versus mayonnaise without additives (negative control); c—mayonnaise with EH mantle versus mayonnaise without additives (negative control); d—mayonnaise with EH muscle versus mayonnaise without additives (negative control), e—mayonnaise with HTE muscle versus mayonnaise without additives (negative control), f—mayonnaise with HTE mantle versus mayonnaise without additives (negative control), and g—mayonnaise with BHT (positive control) versus mayonnaise without additives (negative control) (*p* < 0.05)

The obtained experimental data show that during storage—in addition to the oxidation of the fat phase of mayonnaise—hydrolysis of triglycerides to form free fatty acids is observed: The AV of oil, extracted from the mayonnaise, increases during storage. Acid and enzymatic hydrolysates and hydrothermal extracts in the composition of mayonnaise slow down triglyceride hydrolysis, as evidenced by the decrease of the AV of mayonnaise compared to the control for all investigated samples. The minimum AV throughout the storage period is determined for the fat extracted from the mayonnaise with acid hydrolysate. Acid and enzymatic hydrolysates and hydrothermal extracts in mayonnaise reduce the AV (at 90 days storage) by 20%–44%.

#### **4. Conclusions**

The hydrolysates and hydrothermal extracts of the bivalve mollusk *A. broughtonii* possess antiradical activity, the level of manifestation of which depends on the method of biotechnological and technological processing. Assessment of antiradical activity using the DPPH radical showed that acid hydrolysates have the maximum antiradical properties, probably due to the higher content of low molecular fractions of melanoidins.

Acid and enzymatic hydrolysates, and hydrothermal extracts in the mayonnaise composition slow down the processes of lipid oxidation and hydrolysis of triglycerides. Acid hydrolysates play the most significant role in reducing the speed of oxidation and hydrolysis. The results of this study can be practically applied in the production of mayonnaise and fat-containing sauces. However, the hydrolysates and extracts were not able to prevent the formation of peroxides as is the case for EDTA, which is a much better antioxidant in mayonnaise than BHT. Moreover, peroxides do not have any effect on off-flavors. To make a more complete evaluation of the effect of the hydrolysates and extracts determination of secondary lipid oxidation products such as aldehydes and ketones is necessary and preferably sensory analysis should also be done.

**Author Contributions:** O.V.T. and A.V.T. provided the idea of the research; and carried out the whole experiment (O.V.T. and A.V.T. contributed to this article equally); D.V.P. verified some of the experimental results; T.K.K. and W.P. analyzed and compared the results of the experiment; S.N.M. verified some of the experimental results; O.V.T., W.P., L.P. provided the framework of the paper and finally checked the quality of the article. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Anti-Hyperglycemic E**ff**ects of Green Crab Hydrolysates Derived by Commercially Available Enzymes**

#### **Bouhee Kang 1, Denise I. Skonberg 1,\* and Angela D. Myracle <sup>2</sup>**


Received: 15 January 2020; Accepted: 25 February 2020; Published: 28 February 2020

**Abstract:** The predation and burrowing activity of invasive green crabs have had detrimental effects on important marine resources and habitats. Our objective is to develop bioactive hydrolysates by enzymatic proteolysis of underutilized green crab. Mechanically separated mince was hydrolyzed with Alcalase, Protamex, Flavourzyme, and Papain (1%) for 60 min. Subsequently, the hydrolysates were introduced to a simulated gastrointestinal digestion model. Selected samples were fractionated by ultrafiltration, and their anti-hyperglycemic effects includingα-glucosidase, α-amylase, and dipeptidyl peptidase-IV (DPP-IV) inhibitory activities and glucagon-like 1 (GLP-1) secretory activity were evaluated. The Protamex treatment showed the highest α-glucosidase inhibitory activity (IC50 1.38 ± 0.19 mg/mL) compared to other enzyme treatments and the crab mince control, and its α-amylase inhibitory activity (IC50 11.02 ± 0.69 mg/mL) was lower than its α-glucosidase inhibitory activity. Its GLP-1 secretory activity was approximately four times higher than the positive control (10 mM glutamine). The <3 kD fraction contributed significantly to the anti-hyperglycemic activity of Protamex-derived hydrolysates, and this activity was stable after simulated digestion. Our results suggest that green crab hydrolysates obtained by Protamex treatment have the potential for type 2 diabetes management and could be incorporated in food products as a health-promoting ingredient.

**Keywords:** green crab; protein hydrolysates; enzymatic hydrolysis; type 2 diabetes; anti-hyperglycemia

#### **1. Introduction**

In recent years, consumer interest in sustainability and healthy lifestyles has significantly increased, leading to higher demand for nutritious and environmentally-friendly food products among consumers and the food industry. The development of natural, bioactive food ingredients from currently underutilized food resources may positively contribute to satisfying this demand. Nutritional and bioactive food ingredients include proteins and peptides, polyunsaturated fatty acids (PUFAs), phytochemicals, fibers, probiotics, and prebiotics [1]. Marine organisms represent good sources for obtaining various bioactive compounds, and many of these bioactive compounds are derived from proteins [2]. Therefore, protein-rich marine organisms are an ideal raw starting material for the development of protein-derived bioactive compounds.

European green crabs (*Carcinus maenas*) are an invasive species well established in the U.S. that negatively influence fisheries, aquaculture, and marine habitats due to their high resilience, voracious predation, and strong burrowing activity [3–6]. Green crabs are very competitive predators that prefer juvenile clams, blue mussels, and lobster larvae, which are highly profitable for fisheries and aquaculture, as their prey [3,4]. Their intensive burrowing and high viability have destroyed not only valuable eelgrass beds and salt marshes but also the overall balance of the natural marine ecosystem [5]. Although there is a large population of green crabs in the U.S., they are not currently utilized by food industries due to their small size. To obtain the nutritious meat, significant labor and high costs are required to separate the muscle from the carapace. However, our previous studies demonstrated that green crab mince obtained via mechanical separation can be used in the development of value-added food products [7,8].

Green crab contains various nutritional and nutraceutical compounds such as chitin, carotenoids, essential amino acids, and PUFAs including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [9]. Green crab meat has approximately 80% protein [10]; these proteins have the potential to be utilized as the initial material for the generation of multifunctional food ingredients. Some proteins and peptides extracted from protein-rich marine resources have been reported to act as functional and bioactive ingredients in food products [11,12]. Enzymatic hydrolysis of a variety of protein sources has been extensively utilized as a method to generate bioactive peptides having antioxidant, anti-inflammatory, and anti-hypertensive effects [13–15]. Different types of proteases used for enzymatic hydrolysis have distinct specificity and produce peptides having different molecular weights and amino acid sequences, which may contribute to their diverse biological and physiochemical functional properties [16–18]. Various studies have confirmed that commercially available proteases such as Alcalase, Protamex, Flavourzyme, and Papain have the potential to generate bioactive peptides from food proteins [19,20]. However, the majority of these studies were conducted with dairy proteins and finfish byproducts. Furthermore, the bioactivity of hydrolysates obtained from marine resources was studied extensively with regard to their anti-hypertensive and antioxidant activities rather than their anti-diabetic properties. Fewer studies have been conducted to assess the biofunctional activity of the valuable peptides derived from shellfish proteins. More specifically, there is a lack of research about the potential anti-diabetic activity of crustacean and mollusk proteins and their hydrolysates.

Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic diseases in the world and ranks in the top 10 diseases causing the most deaths worldwide (WHO) [21]. T2DM and associated complications result not only in health issues but also substantial direct and indirect costs [22]. However, an optimal diet can help in the prevention of T2DM and is critical to reducing the attendant morbidity and cost associated with the treatment of T2DM [23–25]. The development of anti-hyperglycemic ingredients from green crabs may contribute to the prevention of T2DM and reduce the use of pharmaceutical products and the risks associated with their side-effects [26]. Furthermore, the development of natural and nutraceutical peptides generated from green crabs may encourage the development of a fishery for this invasive predator. Therefore, the aim of our study was to investigate the anti-hyperglycemic effect of green crab hydrolysates for potential use as a health-promoting food ingredient. To achieve this, (1) enzymatic hydrolysis using commercially available proteases was applied to mechanically separated green crab mince, (2) the potential anti-hyperglycemic effects of green crab hydrolysates were investigated, (3) fractionation of hydrolysates using ultrafiltration was conducted, and (4) changes in anti-hyperglycemic activity of green crab hydrolysates after simulated gastrointestinal digestion were investigated.

#### **2. Materials and Methods**

#### *2.1. Materials and Reagents*

Green crabs (carapace width: 40–85 mm and weight: 20–155 g) were harvested in 2018 on the Back River in Georgetown, Maine, USA. All of the chemicals and reagents that were used in this study were supplied by Fisher Scientific (Hampton, NH, USA), Thermo Fisher Scientific (Waltham, MA, USA), Bio-Rad Laboratories (Hercules, CA, USA), and Sigma-Aldrich (St. Louis, MO, USA) unless otherwise described. Alcalase 2.4 L (AL), Protamex (PR), Flavourzyme (FL), and Papain (PA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rat intestinal acetone powders of

α-glucosidase (EC 3.2.1.20), porcine pancreatic α-amylase (EC 3.2.1.1), pepsin from porcine gastric mucosa (EC 3.4.23.1, >250 units/mg protein), pancreatin from porcine pancreas (EC 232.468.9, 8 × USP), p-nitrophenyl-β-D-glucopyranoside (p-NPG), and Gly-Pro-p-nitroanilide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Porcine kidney dipeptidyl peptidase-IV (DPP-IV) (E.C. 3.4.14.5) and glucagon-like 1 (GLP-1) ELISA kit were purchased from EMD Millipore (Billerica, MA, USA). Cell culture materials including penicillin-streptomycin (0.1g/L), Dulbecco's modified Eagle's medium (DMEM) containing low glucose, Glutamax, trypsin/EDTA (10×), and Poly D-lysine were obtained from Gibco Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Minneapolis, MN, USA). Bovine serum albumin, acarbose, sitagliptin, and glutamine were obtained from Sigma-Aldrich (St. Louis, MO, USA).

#### *2.2. Preparation of Green Crab*

Forty kilograms of harvested crabs were washed with tap water and then blast-frozen at −30 ◦C for 1 h. Frozen crabs were stored at −20 ◦C until further use. Partially thawed green crabs were processed through a mechanical separator (Paoli One-Step mechanical separator, Rockford, IL) to separate meat and shell streams, and then the minced meat was vacuum sealed in plastic bags (3 mil, 3.3 cm3/100 in2 oxygen transmission rate, 80 microns, UltraSource, Kansas, MO, USA). The crab mince was blast-frozen at −30 ◦C for 1 h and then stored at 20 ◦C until further use (Figure 1).

**Figure 1.** Process flow of sample preparation.

#### *2.3. Enzymatic Hydrolysis of Crab Mince*

Enzymatic hydrolysis using commercially available proteases was conducted based on a modified protocol from Beaulieu et al. [27]. The raw crab mince was mixed with deionized water in a 1:1 (*w*/*w*) ratio, then homogenized for 1 min in a Waring blender at maximum speed. The enzymatic hydrolysis was conducted at the optimum pH and temperature of each enzyme including Alcalase (AL, 50 ◦C and pH 8), Protamex (PR, 50 ◦C and pH 7), Flavourzyme (FL, 50 ◦C and pH 7), and Papain (PA, 65 ◦C and pH 6). Protein content was determined based on total nitrogen content as analyzed by a combustion analyzer (TRU MAC CNS, LECO Corp., St. Joseph, MI, USA). Protein content was determined using a protein conversion factor of 6.25. Enzyme was added to the homogenate based on substrate (protein) content (1:100 = E:S), and the mixture was hydrolyzed for 60 min. During hydrolysis, the pH of the mixture was maintained using 6 N HCl or 6 N NaOH and temperature was monitored. After hydrolysis, the mixture was heated at 85–90 ◦C for 10 min to inactivate the enzyme and then immediately cooled on ice. For the crab mince control (CMC), mince was homogenized with water but not subsequently treated with the enzyme. Subsampling was performed to evaluate the degree of hydrolysis, and then the mixtures were centrifuged at 19,722× *g* for 15 min at 4 ◦C, and the supernatants were collected. All of the treatments were processed in triplicate. The collected supernatants were blast-frozen at −30 ◦C for 1 h, then freeze-dried (35 EL, VirTis Co. Inc., Gardiner, NY, USA) at −30 to 25 ◦C under 250 mT for 10 days All lyophilized supernatants were stored at −80 ◦C until further use.

#### *2.4. Degree of Hydrolysis*

Degree of hydrolysis was determined following the O-phthalaldehyde (OPA) method [28,29]. OPA reagent was prepared with 375 mL of deionized water, 19.05 g of sodium tetraborate decahydrate, 500 mg of sodium dodecyl sulfate (SDS), and 400 mg of 97% OPA in 10 mL of ethanol. After mixing, 440 mg of 99% dithiothreitol (DTT) was added to the solution and deionized water was added to achieve a final volume of 500 mL. For the sample preparation, the CMC and enzyme hydrolysates were diluted with 4% SDS (1:19 *w*/*v*). After centrifugation at 1100× *g* for 10 min, 4 mL of supernatant was collected. Subsequently, the supernatant was diluted to 50 mL with deionized water. Four mL of OPA reagent were mixed with 400 μL of solubilized sample/standard (0.5 mg/mL serine) and the mixture was incubated at room temperature for 2 min. Absorbance was measured at 340 nm and the degree of hydrolysis was calculated based on the following three equations:

$$\text{Serine-NH}\_2 \left( \text{mequiv/g protein} \right) = \frac{\frac{\left( \text{OD}\_{\text{sample}} - \text{OD}\_{\text{blank}} \right)}{\left( \text{OD}\_{\text{standard}} - \text{OD}\_{\text{blank}} \right)} \times 0.9516 \frac{\text{mcapiv}}{\text{L}} \times 0.05 \times 100}{\text{W} \times \frac{\text{V}\_1}{\text{V}\_2} \times \text{P}} \tag{1}$$

W = weight of sample (g); P = protein (g/100 g sample); V1 = volume of supernatant (4 mL); V2 = volume of SDS solution (20 mL); 0.05 = sample volume in L.

$$\mathbf{h} = \frac{(\text{Serrine} - \text{NH2}) - \beta}{\alpha} \tag{2}$$

h = number of hydrolyzed peptide bonds; α = 1.00, β = 0.40 mequiv/g protein;

$$\text{DH} = \frac{\text{h}}{\text{h}\_{\text{total}}} \times 100\tag{3}$$

Total number of peptide bonds (htotal) of fish: 8.6 mequiv/g protein.

#### *2.5. Simulated Gastrointestinal Digestion*

Simulated gastrointestinal digestion of the control and crab mince hydrolysates was performed according to González-Montoya et al. [30]. The sample was dissolved in deionized water (5% *w*/*v*), then pH was adjusted to pH 2 using 6 N HCl prior to heating to 37 ◦C. Pepsin was added to the sample mixture (4:100 = E:S protein/peptide content) and stirred at 37 ◦C for 60 min. Then, pH was adjusted to 7.5 using 6 N NaOH. Subsequently, pancreatin (4:100 = E:S protein/peptide content) was added for the intestinal digestion phase and the mixture was stirred at 37 ◦C for 2 h. To inactivate the digestive enzymes, the mixture was heated at 85–90 ◦C for 10 min. The mixture was freeze-dried (35 EL, VirTis Co. Inc., Gardiner, NY, USA) at −30 to 25 ◦C under 250 mT for 10 days and then stored at −80 ◦C until further use.

#### *2.6. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)*

Molecular weight distribution was determined by SDS-PAGE. All of the samples were dissolved in a Laemmli sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 25% (*v*/*v*) glycerol, 0.01% bromophenol blue, and 5% β-mercaptoethanol, then heated in a 90–95 ◦C water bath for 5 min [31]. Samples were separated on a 4% stacking and 16.5% separating gel (Bio-Rad Laboratories, Hercules, CA) using Tris/Glycine/SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% (*w*/*v*) SDS) in a Bio-Rad Mini-PROTEAN III Cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The amount that was loaded to the wells was adjusted to 1 mg for the hydrolysates, 0.5 mg for the control, and 10 μL of a protein molecular weight standard (Precision Plus Protein™ Dual Xtra Prestained Protein Standards 2-250 kD, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Separated proteins were fixed in a gel fixing solution containing 50% (*v*/*v*) ethanol and 10% (*v*/*v*) acetic acid then washed with a solution consisting of 50% (*v*/*v*) methanol and 10% (*v*/*v*) acetic acid. Following washing, gel staining was performed using Coomassie blue R-250 solution at room temperature for 3 h with gentle agitation. (Bio-Rad Laboratories, Inc., Hertfordshire, UK), then the gel was destained with washing solution until the background color was removed.

#### *2.7. Fractionation Using Ultrafiltration*

Green crab hydrolysates were fractionated using ultrafiltration based on molecular weight. A volume of 15 mL of solubilized sample (50 mg/mL) was added into a 30 kD molecular weight cut-off (MWCO) filter device (Amicon® ultra-15 centrifugal filters, EMD Millipore, Burlington, MA. USA) for the first fraction, then centrifuged at 3234× *g* until the volume of retentate reached 250 μL. After collecting the retentate, the <30 kD filtrate was transferred to a 10 kD MWCO filter device for the second fraction. After centrifugation at 3234× *g*, 250 μL of retentate was collected. The <10 kD filtrate was added to the 3 kD MWCO filter device for the third fractionation and was centrifuged at 3234× *g* until the retentate volume reached 250 μL. Both the retentate and the <3 kD fraction were collected and all the hydrolysate fractions were stored at −80 ◦C until used for further assays.

#### *2.8. Rat Intestine* α*-Glucosidase Inhibition Assay*

Rat intestine α-glucosidase inhibition assay was conducted according to the protocol in the Worthington Enzyme Manual with modifications [32] and Kwon et al. [33]. Crude enzyme was extracted from rat intestine acetone powder. For the extraction, 0.3 g of rat intestinal acetone powder was added to 12 mL of 0.1 M sodium phosphate buffer (pH 6.9 with 0.9% NaCl), then sonicated 12 times in 30 s pulses. After centrifugation at 10,000× *g* for 30 min at 4 ◦C, the supernatant was used as the enzyme solution. A volume of 50 μL of solubilized sample or acarbose (positive control) and 100 μL of enzyme solution were added in a 96 well plate then incubated at 37 ◦C for 10 min. Then, 50 μL of 5 mM p-NPG solution in 0.1 M phosphate buffer (pH 6.9 with 0.9% NaCl) was added and the mixture was incubated at 37 ◦C for 30 min. The absorbance was measured at 405 nm by a microplate reader (Ex 808, Biotek, Winooski, VT, USA) and compared with a control containing 50 μL of 0.1 M

sodium phosphate buffer in place of the sample. The α-glucosidase inhibitory activity was calculated as follows:

$$\% \text{ inhibition} = \frac{\left(\Delta \text{A}\_{405}^{\text{Control}} - \Delta \text{A}\_{405}^{\text{sample}}\right)}{\Delta \text{A}\_{405}^{\text{Control}}} \times 100 \tag{4}$$

#### *2.9. Porcine Pancreatic* α*-Amylase Inhibition Assay*

The α-amylase inhibitory activity was evaluated following a modified version of the assay described by the Worthington Enzyme Manual [34] and Kwon et al. [26]. Porcine pancreatic α-amylase solution (100 unit/mL) was prepared in 0.02 M sodium phosphate buffer (pH 6.9 with 6 mM NaCl). A volume of 100 μL of solubilized sample or acarbose (positive control) and 100 μL of enzyme solution was incubated at 25 ◦C for 10 min, then 100 μL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 6 mM NaCl) was added. After incubation at 25 ◦C for 10 min, 200 μL of dinitrosalicylic acid color reagent was added, and, subsequently, the mixture was boiled in a water bath for 5 min. After cooling, 50 μL of the mixture was diluted with 200 μL of deionized water and the absorbance was measured at 540 nm by a microplate reader (Ex 808, Biotek, Winooski, VT, USA). The measured absorbance was compared with a control containing 100 μL of 0.02 M sodium phosphate buffer in place of the sample and the α-amylase inhibitory activity was calculated as follows:

$$\% \text{ inhibition} = \frac{\left(\Delta \text{A}\_{540}^{\text{Control}} - \Delta \text{A}\_{540}^{\text{sample}}\right)}{\Delta \text{A}\_{540}^{\text{Control}}} \times 100 \tag{5}$$

#### *2.10. DPP-IV Inhibition Assay*

The DPP-IV inhibitory activity was determined using a method from Li-Chan et al. [35]. DPP-IV enzyme was purchased from EMD Millipore (Burlington, MA, USA) and 0.01 unit/mL of enzyme solution was prepared in 100 mM Tris-HCl buffer (pH 8.0). A total of 25 μL of sample and sitagliptin (positive control) and 25 μL of 1.59 mM Gly-Pro-p-nitroanilide solution in 100 mM Tris-HCl buffer (pH 8.0) was preincubated at 37 ◦C for 10 min, then 50 μL of enzyme solution was added. After incubation at 37 ◦C for 60 min, 100 μL of 1 M sodium acetate solution was added to stop the reaction. The absorbance was determined at 405 nm by a microplate reader (Ex 808, Biotek, Winooski, VT, USA). The absorbance was compared with a control of 25 μL of 100 mM Tris-HCl buffer (pH 8.0) instead of the sample solution. The DPP-IV inhibitory activity was expressed as percentage inhibition and was calculated as follows:

$$\% \text{ inhibition} = \frac{\left(\Delta \text{A}\_{405}^{\text{Control}} - \Delta \text{A}\_{405}^{\text{sample}}\right)}{\Delta \text{A}\_{405}^{\text{Control}}} \times 100 \tag{6}$$

#### *2.11. GLUTag Cell Culture*

Murine GLUTag cells that are widely used for the stimulation of GLP-1 secretion were used in this study. GLUTag cells were gifted from Dr. D.J. Drucker (University of Toronto, Toronto, Canada) and cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 5.5 mM glucose (pH 7.4) supplemented with 10% (*v*/*v*) fetal bovine serum, 100 U/mL penicillin-streptomycin, and 2 mM L-glutamine in a 75 cm<sup>2</sup> culture flask at 5% CO2 and 37 ◦C and GLUTag cells at 80% confluency were regularly trypsinized for subculture.

#### *2.12. Cell Viability Assay*

Viability of the GLUTag cells was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide) assay. The surface of 96-well culture plates was coated with poly-D-lysine solution. A density of 2 <sup>×</sup> 104 cells was seeded in each well and incubated at 37 ◦C, 5% CO2, and 80% humidity for 48 h. A volume of 20 μL sample solution (final concentration: 0.2–10 mg/mL)

was added to the cells and incubated for 24 h. Then, the mixture was replaced with 100 μL of 0.5 mg/mL MTT reagent and incubated at 37 ◦C and 5% CO2 for 4 h. The MTT reagent was removed, then the precipitate was solubilized in 100 μL of DMSO. The absorbance was measured at 540 nm by a microplate reader (Ex 808, Biotek, Winooski, VT, USA) and compared with a control containing 20 μL of ultrapure water in place of the sample solution. The cell viability was calculated as follows:

$$\% \text{ cell viability} = \frac{\left(\Delta \text{A}\_{540}^{\text{Sample}} - \Delta \text{A}\_{540}^{\text{blank}}\right)}{\left(\Delta \text{A}\_{540}^{\text{Control}} - \Delta \text{A}\_{540}^{\text{blank}}\right)} \times 100 \tag{7}$$

#### *2.13. GLP-1 Secretion Assay*

GLP-1 secretory activity was evaluated by an assay modified from Ojo et al. [36]. GLUTag cells were seeded at a density of 2 <sup>×</sup> <sup>10</sup><sup>5</sup> in 24-well culture plates coated with poly-D-lysine and grown for 48 h in a 37 ◦C and 5% CO2 incubator until the confluency reached 80%. The cells were washed with phosphate-buffered saline (PBS), then 1 mL of Kreb Ringer Buffer (KRB) containing 115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM HEPES, and 1M NaHCO3 supplemented with 1% bovine serum albumin (BSA, fatty-acid-free) and 1 mM glucose (pH 7.4) was added. After pre-incubation at 37 ◦C and 5% CO2 for 45 min, the KRB was replaced with 1 mL of appropriate KRB test solutions containing sample or 10 mM L-glutamine (positive control). All the KRB test solutions were prepared with supplementation of 1% BSA and 2 mM glucose (pH 7.4). The culture plates were incubated at 37 ◦C and 5% CO2 for 2 h, then the mixture was collected. After centrifugation at 4 ◦C and 800× *g* for 5 min, the supernatant was collected and stored at −80 ◦C until further evaluation of GLP-1 concentration. Total GLP-1 concentration was determined based on the manufacturer's instructions in a commercial ELISA kit (GLP-1 Total ELISA, Millipore, Burlington, MA, USA). The GLP-1 secretory activity was expressed as a percentage (%) of the negative control (KRB buffer).

#### *2.14. Statistical Analysis*

The enzymatic hydrolysis process using each of the four commercial proteases was replicated three times and all of the assays were conducted in triplicate on each sample replicate. Statistical differences among the means of each treatment were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's HSD post hoc test and paired *t*-test with a significance value of *p* < 0.05 (SPSS ver. 23, IBM Corp., Armonk, NY, USA). Correlations (*p* < 0.05) between the degree of hydrolysis (DH) and each biofunctional activity were analyzed through the Pearson coefficient (SPSS ver. 23, IBM Corp., Armonk, NY, USA).

#### **3. Results**

#### *3.1. Degree of Hydrolysis*

The degree of hydrolysis (DH) was determined by the OPA method with serine as a standard. DH was significantly increased after enzymatic hydrolysis for 60 min. DH of green crab hydrolysates ranged from 15.8% to 18.4%, while DH of the CMC (crab mince control) was 6.8%. Among the treatments, AL showed the highest DH (18.4% ± 0.4%) followed by the PR (17.1% ± 0.2%), FL (16.5% ± 1.2%), and PA (15.8% ± 0.3%) treatments, respectively (Figure 2).

**Figure 2.** Degree of hydrolysis of green crab mince by enzyme type. CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation. Different letters on the bars indicate a significant difference (*p* < 0.05) among the treatments (*n* = 3).

#### *3.2. Molecular Weight Distribution Using SDS-PAGE*

The size distribution of proteins and peptides in the CMC and hydrolysates was determined using SDS-PAGE (Figure 3a). Significant hydrolysis was observed in all of the treatments compared to the CMC. Heavy chains in the 150–250 kD range were reduced by all the enzyme treatments. There were thick bands between 100 kD and 75 kD in the hydrolysates derived by FL and PA, and all of the treatments produced various bands between 20 kD and 37 kD. Similar band patterns were observed in the AL and PR treatments while FL and PA treatments resulted in similar molecular weight profiles. AL-derived hydrolysate showed the lowest intensity of bands between 2 and 10 kD compared to the CMC and other hydrolysates. During simulated digestion, the CMC was hydrolyzed by digestive enzymes as compared to the process control that was applied to the simulated digestion with no digestive enzymes (Figure 3b). The simulated digestion process further hydrolyzed all the hydrolysates obtained from commercial enzyme treatments. The intensity of bands above 15 kD decreased significantly as compared to before the simulated digestion, and the intensity of observed bands at <10 kD also decreased after the simulated digestion.

#### *3.3.* α*-Glucosidase and Porcine* α*-Amylase Inhibitory Activities*

The inhibitory activity of rat intestinal α-glucosidase and porcine pancreatic α-glucosidase was assessed to evaluate the potential anti-hyperglycemic effect of the CMC and crab hydrolysates. IC50 values of both α-glucosidase and α-amylase were determined to evaluate the efficacy of the inhibition. Acarbose was used as a positive control. Acarbose had IC50 values of 0.027 mg/mL and 0.84 mg/mL for α-glucosidase and α-glucosidase inhibitory activity, respectively. The α-glucosidase inhibitory activity of the CMC was significantly improved by the PR (IC50 1.38 ± 0.19 mg/mL) and PA (IC50 5.56 ± 0.19 mg/mL) treatments while the FL (IC50 20.24 ± 0.19 mg/mL) treatment showed a decrease in α-glucosidase inhibitory activity compared to the CMC (IC50 8.54 ± 0.50 mg/mL) (Figure 4a). Simulated digestion did not significantly affect the α-glucosidase inhibitory activity of hydrolysates obtained from the PR (IC50 1.49 ± 0.08 mg/mL) and PA (IC50 5.31 ± 0.51 mg/mL) treatments. However, the inhibitory activity of the FL-derived hydrolysate was significantly improved after the simulated digestion (IC50 10.61 ± 1.90 mg/mL). The green crab hydrolysates obtained from PR showed the highest α-glucosidase inhibitory activity before and after simulated digestion and the activity was approximately 4–14 times higher compared to other enzyme treatments and CMC based on IC50 values.

**Figure 3.** Molecular weight distribution (SDS-PAGE) of CMC and hydrolysates before and after simulated digestion. (**a**) Before simulated digestion. Lane 1 and 7: molecular weight ladder. Lanes 2–6: CMC, AL-derived hydrolysate, PR-derived hydrolysate, FL-derived hydrolysate, and PA-derived hydrolysate, respectively. (**b**) After simulated digestion. Lanes 1 and 8: molecular weight ladder. Lanes 2–7: simulated digestion process control, CMC, AL-derived hydrolysate, PR-derived hydrolysate, FL-derived hydrolysate, and PA-derived hydrolysate, respectively. CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain.

The porcine pancreatic α-amylase inhibitory activity of the CMC was statistically increased by enzymatic hydrolysis using commercial enzymes. Among the treatments, PA (IC50 9.35 ± 0.42 mg/mL) exhibited the highest α-amylase inhibitory activity followed by PR (IC50 11.02 ± 0.69 mg/mL), FL (IC50 11.12 ± 0.37 mg/mL), AL (IC50 12.53 ± 0.85 mg/mL), and the CMC (IC50 16.49 ± 0.41 mg/mL), respectively (Figure 4b). Digestive enzymes including pepsin and pancreatin significantly improved the α-amylase inhibitory activity of CMC (IC50 10.55 ± 0.68 mg/mL) and its hydrolysates treated with AL (IC50 10.11 ± 0.34 mg/mL), PR (IC50 9.93 ± 0.82 mg/mL), and FL (IC50 9.31 ± 0.37 mg/mL), however, the inhibitory activity of PA-derived hydrolysate was not affected by simulated digestion. After simulated digestion, the α-amylase inhibitory activity among the treatments was not significantly different.

**Figure 4.** Rat intestinal α-glucosidase and porcine α-amylase inhibitory activities of CMC and hydrolysates before and after simulated digestion. (**a**) Rat intestinal α-glucosidase inhibitory activity. (**b**) Porcine α-amylase inhibitory activity. The IC50 values were represented by final assay concentration (protein and peptide basis). CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). The letters on the bars indicate a significant difference (*p* < 0.05) among the treatments. An asterisk (\*) represents significant difference after simulated digestion (*p* < 0.05) by paired *t*-test.

#### *3.4. DPP-IV Inhibitory Activity*

Overall, DPP-IV inhibition of CMC (IC50 1.50 ± 0.25 mg/mL) was significantly improved by the enzymatic hydrolysis, and the DPP-IV inhibitory activity IC50 values of hydrolysates ranged from 0.56 mg/mL to 0.72 mg/mL (Figure 5). Sitagliptin was used as a positive control, and had an IC50 value of 43.7 ng/mL. The simulated gastrointestinal digestion remarkably enhanced the DPP-IV inhibitory activity of CMC. However, the PR and Papain-treated samples showed significantly higher DPP-IV inhibitory activity than CMC after the simulated digestion.

**Figure 5.** Dipeptidyl peptidase-IV (DPP-IV) IC50 values of CMC and hydrolysates. The IC50 values were represented by the final assay concentration (protein and peptide basis). CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). The letters on the bars indicate a significant difference (*p* < 0.05) among the treatments. An asterisk (\*) represents a significant difference after simulated digestion (*p* < 0.05) by paired *t*-test.

#### *3.5. GLP-1 Secretory Activity*

The GLP-1 secretory activity was measured at a sample concentration of 0.5 mg/mL. The cytotoxicity of the CMC and hydrolysates was determined using the MTT assay before evaluating their GLP-1 secretory activity. As a result, there was no observed cytotoxicity on GLUTag cells at 0.125, 0.25, and 0.5 mg/mL (Figure S1). The GLP-1 levels induced by the CMC and crab hydrolysate treatments were evaluated using the GLUTag cell model. Next, the results were compared with glutamine as a positive control (Figure 6). The GLP-1 secretion induced by the CMC and hydrolysates was significantly higher than that by the positive control by 2.5–3.5 times. Among the samples, AL-derived hydrolysate showed the lowest GLP-1 secretory activity, and the activity of all the samples was not significantly affected by simulated gastrointestinal digestion.

**Figure 6.** Glucagon-like 1 (GLP-1) level of CMC and hydrolysates before and after simulated digestion determined using GLUTag cells. The GLP-1 level was measured using 0.50 mg/mL of samples (final assay concentration, protein, and peptide basis). CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). The letters on the bars indicate a significant difference (*p* < 0.05) among the treatments.

#### *3.6.* α*-Glucosidase Inhibitory Activity of Fractions*

Based on the positive results of their α-glucosidase and GLP-1 secretory activities, PR, FL, and PA were selected for subsequent fractionation. Figure 7 represents the α-glucosidase inhibitory activity of the fractions determined at 3.2 mg/mL. PR-treated fractions exhibited the highest α-glucosidase inhibitory activity among the treatments. Within the fractions, the highest α-glucosidase inhibitory activity was shown in the <3 kD fractions of all of the treatments. The <3 kD fraction of the PR-derived hydrolysate inhibited the α-glucosidase activity by 60% while the α-glucosidase inhibitory activity of other fractions was less than 50%. IC50 values could only be calculated for the <3 kD fraction since the highest inhibitory activity of the >3 kD fractions was less than 50%. The PR treatment (IC50 1.75 ± 0.24 mg/mL) resulted in the highest α-glucosidase inhibitory effect followed by PA, CMC, and FL.

**Figure 7.** Rat intestinal α-glucosidase inhibitory activities of CMC and hydrolysates after fractionation (final assay concentration: 3.2 mg/mL protein and peptide basis). CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). Uppercase letters on the bars indicate a significant difference (*p* < 0.05) within each treatment. Lowercase letters on the bars indicate a significant difference (*p* < 0.05) among the treatments.

#### *3.7.* α*-Amylase Inhibitory Activity of Fractions*

The α-amylase inhibition of all the fractions was evaluated at 4.2 mg/mL (Figure 8). The <3 kD fractions had the highest inhibitory activity among the fractions. The inhibitory activities of the <3 kD fractions were not significantly different between PR and FL treatments, which were significantly higher than the α-amylase inhibitory activities of the <3 kD fractions of CMC and PA-derived hydrolysates. IC50 values of all the <3 kD fractions ranged from 8.36 to 10.88 mg/mL and were not significantly different among the treatments.

#### *3.8. DPP-IV Inhibitory Activity of Fractions*

The DPP-IV inhibitory ability of the fractions obtained from the fractionation of the ±CMC and hydrolysates is shown in Figure 9. According to IC50 values, the <3 kD fraction (CMC: 1.39±0.16 mg/mL, PR: 0.79 ± 0.05 mg/mL, FL: 0.88 ± 0.17 mg/mL, and PA: 0.93 ± 0.20 mg/mL) and 3–10 kD fraction (CMC: 1.68 ± 0.28 mg/mL, PR: 0.95 ± 0.06 mg/mL, FL: 1.15 ± 0.16 mg/mL, and PA:0.88 ± 0.17 mg/mL) within each sample showed the highest DPP-IV inhibitory activity while the > 30 kD fraction (CMC: 4.80 ± 0.37 mg/mL, PR: 2.29 ± 0.40 mg/mL, FL: 2.70 ± 0.26 mg/mL, and PA: 2.21 ± 0.20 mg/mL) exhibited the lowest DPP-IV inhibitory activity within enzyme treatments. The DPP-IV inhibitory activity of all

the fractions was improved after the commercial enzyme treatments; however, there was no significant difference among the treatments.

**Figure 8.** Porcine pancreatic α-amylase inhibitory activities of CMC and hydrolysates after fractionation (final assay concentration: 4.2 mg/mL protein and peptide basis). CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). Uppercase letters on the bars indicate a significant difference (*p* < 0.05) within each treatment. Lowercase letters on the bars indicate a significant difference (*p* < 0.05) among the treatments.

**Figure 9.** DPP-IV IC50 values of CMC and hydrolysates after fractionation. The IC50 values were represented by final assay concentration (protein and peptide basis). CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). Uppercase letters on the bars indicate a significant difference (*p* < 0.05) within each treatment. Lowercase letters on the bars indicate a significant difference (*p* < 0.05) among the treatments.

#### *3.9. GLP-1 Secretory Activity of Fractions*

After the fractionation, the total GLP-1 level induced by the CMC and crab hydrolysates (0.5 mg/mL) was evaluated using GLUTag cells. The GLP-1 secretory activity of fractions was improved by the enzymatic hydrolysis except for the >30 kD fraction of PA-derived hydrolysate and <3 kD fractions of

FL and PA-derived hydrolysates (Figure 10). Glutamine (10 mM, positive control) increased GLP-1 level by 216% ± 13% compared to a negative control (KRB buffer), and the <3 kD fractions had significantly higher GLP-1 levels than the positive control.

**Figure 10.** GLP-1 level of CMC and hydrolysates after fractionation (final assay concentration: 0.5 mg/mL protein and peptide basis). CMC: Crab Mince Control, AL: Alcalase, PR: Protamex, FL: Flavourzyme, and PA: Papain. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). Lowercase, lowercase letters with underline, and Greek letters on the bars indicate a significant difference (*p* < 0.05) within each treatment. Uppercase letters on the bars indicate a significant difference (*p* < 0.05) among the treatments.

#### **4. Discussion**

In recent years, as interest in healthy lifestyles has increased, the demand for healthy diets, natural food ingredients, and bioactive compounds has intensified in consumers as well as the food industry. Proteins and peptides are being widely used as nutraceutical food ingredients in food products and dietary supplements to enhance human health and product value. Many bioactive peptides having antioxidant, anti-hypertensive, and anti-hyperglycemic effects have been derived from enzymatic hydrolysis of protein sources including meat, dairy, fish, and their byproducts [15–20,37–39]. However, there has been limited investigation into bioactive peptides derived from molluscan and crustacean resources.

The bioactive properties of peptides obtained by enzymatic hydrolysis are influenced by (1) the primary amino acid sequence of the protein source, (2) the specificity of the proteases applied, and (3) process parameters such as DH, hydrolysis duration, and enzyme:substrate ratio. As a primary protein source, underutilized green crab was used, which contains ~80% protein in its meat [7]. Manual removal of green crab meat from the carapace is extremely labor-intensive because of the crab's very small size. Therefore, in this study, a mechanical separator was used to generate green crab meat mince that was subsequently used as the substrate for the enzymatic hydrolysis. To apply proteases with various specificity to the crab homogenate, four commercially available proteases that have two different modes of action were selected. AL, PR, and PA are endopeptidases, while FL has both endoand exopeptidase characteristics. After hydrolysis, the DH was determined based on the number of cleaved peptide bonds over the total number of peptide bonds. Based on our results, DH significantly increased in response to enzyme treatment (Figure 2). According to Jamdar et al. [40], the bioactivity of protein hydrolysates was modified as DH was changed within the same enzyme treatment. Therefore,

the determination of DH is necessary for controlling the enzymatic hydrolysis process to reproducibly obtain bioactive peptides.

In addition to the increase in DH, the molecular weight distribution of the CMC and hydrolysates before the simulated digestion also confirms that the enzymatic process was successful, and that different products were generated based on the commercial protease applied (Figures 2 and 3). Although similar MW patterns were observed in AL and PR-treated samples, their activities were significantly different in terms of α-glucosidase inhibition and GLP-1 secretion (Figures 3 and 5). The weak intensity of AL-derived hydrolysate bands between 2 and 10 kD may be associated with its DH (Figure 3a). In many studies, AL showed an outstanding ability to hydrolyze large proteins to small peptides, and produced smaller peptides more rapidly compared to other commercially available proteases [41,42]. Therefore, the lower intensity of the 2–10 kD bands in the AL treatment was most likely due to a large number of peptides smaller than <2 kD passing through the gel during the SDS-PAGE. After the simulated digestion, the amount of larger size peptides was decreased, and the overall intensity of all the bands became weaker (Figure 3a,b). This demonstrates that proteins and peptides in the CMC and hydrolysates were further hydrolyzed by the digestive enzymes and suggests that the smaller sized peptides may have passed through the gel during electrophoresis.

Pancreatic α-amylase and intestinal α-glucosidase are the two key enzymes involved in starch digestion, resulting in an increase in blood glucose levels. Therefore, the inhibition of both carbohydrases can be an indicator of the potential anti-hyperglycemic effect [43]. According to the DH and SDS-PAGE results, AL hydrolyzed the green crab protein to a greater degree compared to the other enzymes (Figures 2 and 3a). However, results indicate that PR generated the most effective α-glucosidase inhibitive hydrolysates (Figure 4), and that the inhibitory activity was not statistically correlated (*p* > 0.05) with DH. The IC50 values of the control and the hydrolysates were strongly correlated with the <3 kD fractions (*r* = 0.987, *p* < 0.05), which indicates that the peptides in the <3 kD fraction are key contributors to the α-glucosidase inhibitory activity. The α-glucosidase inhibitory activity of PR-treated sample (IC50 1.38 ± 0.19 mg/mL) was remarkably higher than not only other treatments and the control but also the hydrolysates of sardine muscle (IC50 48.7 mg/mL), whey protein isolate (IC50 4.5 mg/mL), and edible insects (IC50 > 2.0 mg/mL) including mealworm larvae, crickets, and silkworm pupae [38,44,45]. In many studies, AL, FL, and digestive enzymes including pepsin and trypsin are commonly used to obtain bioactive peptides [38,39,45,46]. Interestingly, in the current study, AL and FL treatment did not show improved inhibitory activity compared to the control. The high α-glucosidase inhibitory activity of the PR-treated sample emphasizes the importance of protein source and protease selection in obtaining bioactive peptides. A major concern regarding the use of bioactive peptides for human consumption and industrial application are the changes in biological activity as a result of further hydrolysis during gastrointestinal digestion and processing. However, simulated digestion did not affect the α-glucosidase inhibitory activities of PR and PA-treated samples, suggesting that their inhibitory activity was stable to pepsin and pancreatin action during the gastrointestinal digestion.

The enzymatic hydrolysis by commercial enzymes, and subsequently by digestive enzymes, improved the α-amylase inhibitory activity of the green crab proteins. The inhibitory activity was most likely primarily due to the <3 kD fractions (Figures 4b and 8) since those showed the highest inhibitory activity among the same concentration of fractions. The α-amylase inhibition of the PR-treated sample was weaker than its α-glucosidase inhibition which may contribute to reducing the side-effects that are frequently caused by inhibition of carbohydrate hydrolyzing enzymes. Acarbose, a synthetic pharmaceutical α-glucosidase and α-amylase inhibitor, has been commonly used to inhibit glucose absorption in diabetic patients. However, its strong α-amylase inhibitory activity (IC50 <1 mg/mL) leads to the presence of non-digested polysaccharides in the large intestine, which causes side-effects including severe stomach pain, constipation, and diarrhea [47]. Therefore, the use of α-glucosidase and α-amylase inhibitors based on the PR-derived hydrolysates might be a good alternative to delay glucose absorption and help control blood glucose spikes. The inhibitory mechanism of both α-glucosidase and α-amylase by bioactive peptides has not been well characterized. However, recent studies have

reported that hydrophobic interactions of non-saccharide compounds that allow them to bind to the carbohydrase active site may contribute to their inhibitory activity [48].

DPP-IV is an enzyme that rapidly metabolizes incretins such as active GLP-1 and gastric inhibitory polypeptide (GIP) hormones. These incretins are important since the hormones help in blood glucose control by insulin secretion [49]. DPP-IV as a postproline hydrolyzing enzyme cleaves dipeptides with X-Pro or X-Ala from the N-terminus of polypeptides [50,51]. In this study, the DPP-IV inhibitory activity of the CMC and hydrolysates was investigated as a potential strategy for T2DM management. Enzyme hydrolysis improved the DPP-IV inhibitory activity regardless of the protease applied, and the activity was not significantly (*p* > 0.05) correlated with DH. Recent studies on DPP-IV inhibitors derived from food sources reported that peptides containing Pro, Ala, and Gly at the P1-position and/or Trp at the N-terminal might have effective DPP-IV inhibitory activity [52]. The type of amino acid residues at the P1, P2, and P1 -positions of peptides may significantly influence their DPP-IV inhibitory activity [53]. The <3 kD and 3–10 kD fractions showed similar IC50 values to the hydrolysates before the simulated digestion (Figures 5 and 9). Thus, our study suggests that the fractions <10 kD are likely to play an important role in inhibiting the DPP-IV enzyme. However, overall DPP-IV inhibitory activity of the CMC and hydrolysates (IC50 0.56–1.5 mg/mL) was significantly lower than the medication sitagliptin (IC50 43.7 ng/mL) and other dairy protein and salmon byproduct hydrolysates (IC50 < 100 μg/mL) [35,38].

GLP-1 hormone has the ability to reduce blood sugar levels by enhancing the secretion of insulin; however, it has a half-life of only ~1.5 min because of rapid inactivation by DPP-IV [54]. The GLP-1 content secreted from GLUTag cells was not different among the samples except for AL treatment that showed a lower GLP-1 level, and the GLP-1 secretory activity was not correlated with DH. In comparison with the positive control and hydrolysates from blue whiting and salmon skin, green crab hydrolysates released approximately 2.5–3.5 times higher GLP-1 levels. This is considered due to the mixture of certain free amino acids and peptides in the crab hydrolysates. Various amino acids including Gln, Glu, Ala, Ser, Leu, Gly, Asn, and Met have been demonstrated to stimulate GLP-1 secretory activity [39,55–58]. According to Tolhurst [59], there are two major mechanisms involved in the stimulation of GLP-1 release by Gln on Glutag cells: (1) electrogenic sodium-coupled amino acid uptake resulting in a depolarization of membrane and activation of voltage-gated calcium entry, and (2) elaboration of intracellular cAMP levels. Gameiro et al. [58]. reported that amino acids such as Ala and Gly activate ionotropic glycine receptors, which respond to the various amino acids by generating chloride current. Our previous research showed that the green crab mince contains a high amount of Gln + Glu and Asn + Asp [7], which may have contributed to its higher GLP-1 secretory activity compared to the negative and positive controls. Small peptides including tri- and dipeptides (Leu-Gly-Gly, Gly-Leu, and Gly- Pro) are known to stimulate the secretion of GLP-1 from the in vitro cell model [39,60]. Therefore, the high GLP-1 content induced by the <3 kD fractions within treatments might be due to the concentration of amino acids and small di- or tripeptides after the fractionation.

#### **5. Conclusions**

Enzymatic hydrolysis was successfully applied to mechanically separated green crab mince to generate a potential anti-hyperglycemic food ingredient. Our results suggest that the anti-hyperglycemic effects of green crab hydrolysates were dependent on the type of protease applied, and not on the degree of hydrolysis. Among the proteases evaluated, Protamex generated products having the highest α-glucosidase inhibitory activity. Furthermore, the fractionation study indicated that the <3 kD peptides primarily contributed to the bioactivities of the hydrolysates. Importantly for their prospective use in novel food products, the bioactivities of the hydrolysates were stable after the simulated gastrointestinal digestion. In conclusion, Protamex was the most effective protease for obtaining anti-hyperglycemic hydrolysates from green crab mince, and the development of bioactive hydrolysates may be a viable route for developing value-added food ingredients from this underutilized marine resource. However, substantial further research including identifying the bioactive compounds in the hydrolysates, evaluating their stability in a food model, and conducting in vivo assessment of their effectiveness is required for their future commercial application in health-promoting foods.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/3/258/s1, Figure S1: Effect of CMC and hydrolysates on GLUTag cells. Each bar indicates the mean and standard deviation (*n* = 3 treatment replicates). Asterisk (\*) represents significant difference between negative control and each sample (*p* < 0.05) by paired *t*-test.

**Author Contributions:** Conceptualization, B.K., D.I.S., and A.D.M.; methodology, B.K., D.I.S., and A.D.M.; validation, B.K., D.I.S., and A.D.M.; formal analysis, B.K.; investigation, B.K.; data curation, B.K.; writing—original draft preparation, B.K.; writing—review and editing, D.I.S., and A.D.M.; supervision, D.I.S., and A.D.M.; funding acquisition, D.I.S., and A.D.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by National Science Foundation award, grant number 1355457 to Maine EPSCOR at the University of Maine.

**Acknowledgments:** We are thankful to Marissa McMahan at Manomet (Brunswick, ME) for providing green crabs. This manuscript is #3730 of the University of Maine Agricultural and Forest Experiment Station.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


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