1 Introduction

Marine bivalves live under the highest microbial concentrations in seawater, approximately 106 bacteria/ml and 109 virus/ml of seawater (Diaz 2010). So, they need to have vigorous immune system strategies. The antimicrobial peptides (AMPs) constitute the first line of these defense strategies against invading microorganisms to survive in this marine environment (Falanga et al. 2016; Zannella et al. 2017). Marine bivalves are considered one such rich source of these AMPs that possess various biological activities, such as antibacterial, antioxidant, anticoagulant, etc. (Galdiero et al. 2015). The AMPs show antimicrobial properties, and provide a rapid and immediate response against the invading microbes (Boman 1995; Bartlett et al. 2002). They are small peptides, less than 60 amino acids and less than 10 kDa in mass, and are triggered immediately after microbial infection. Their value in innate immunity lies on their ability to function without either high specificity or memory. Also, they can be synthesized without dedicated cells or tissues, and they rapidly diffuse to the point of infection (Relf et al. 1999; Chandran et al. 2009). AMPs are effective against dormant bacteria that can survive in high concentrations of antibiotics and need an extensive treatment (Lai and Gallo 2009; Hurdle et al. 2011). Also, AMPs have remarkable specificity for prokaryotes, such as bacterial pathogens without cytotoxicity to eukaryotic host cells (Matsuzaki 2009).

These AMPs are characterized by their high cysteine content, and they have been organized into four groups according to their primary structure, especially cysteine array; defensins, mytilins, myticins and mytimycin (Parisi et al. 2009). Despite variations in structure and size of AMPs, most of them have cationic and amphiphilic characters, and possess affinity towards both hydrophilic and hydrophobic surfaces. These peptides generally act by forming pores in microbial membranes disrupting membrane integrity (Tam et al. 2000). Microbes can not develope resistance against this action mode (Falanga et al. 2016; Zannella et al. 2017).

Moreover, some AMPs can be effective in inhibiting viral infections by many mechanisms, including prevention of viral attachment or penetration into the host cells by interaction with the specific cellular receptor, blocking early steps of viral entry by surface carbohydrate interaction, inactivation of viral envelope glycoproteins, inhibition of viral DNA and/or RNA synthesis, blocking intracellular expression of viral genes and/or production of viral proteins and modulation of antiviral responses of host cell (Dang et al. 2015; Zannella et al. 2017).

Marine bivalves Cerastoderma glaucum (Bruguière 1789) survive in a hostile environment in Lake Timsah, Egypt, where they are surrounded by different pathogenic organisms, including human pathogens. So, the present study was conducted to evaluate and compare the efficacy of whole-body soft tissue extracts of the marine bivalve C. glaucum using different types of polar and nonpolar solvents as an antimicrobial. Further study is extended to detect the antimicrobial substances of C. glaucum using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform-infrared (FT-IR).

2 Materials and methods

2.1 Study site and sample collection

Specimens of Cerastoderma glaucum (the common name is cockle) were collected during winter and summer, 2018 from Lake Timsah, Ismailia, Egypt. This lake is small and shallow, situated between 30° 33′3′′ and 30° 35′ 31′′ North latitude and 32° 16′ 30′′ and 32° 18′ 50′′ East longitude (Ibrahim and El-Regal 2014; Kandeel 2018) (Fig. 1). The whole-body soft tissues of cockle samples (31.58 ± 0.485 mm long, 18.67 ± 0.334 mm width and 25.02 ± 0.278 mm height) were collected in polyethylene bags and freeze-dried as a whole in isothermal boxes.

Fig. 1
figure 1

Map of Lake Timsah, Ismailia, Egypt (Kandeel 2018)

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2.2 Preparation of the tissue extracts of Cerastoderma glaucum

Three different solvents, chloroform, methanol and hydrochloric acid, were used. The chloroform and methanol extracts were prepared by keeping 150 g of the homogenated tissues in 150 ml of these solvents overnight at 4 °C and then centrifugation at 10,733 × g for 20 min. The supernatant was concentrated using a vacuum rotary evaporator (35–55 °C) to give predominantly an aqueous suspension which was lyophilized to give yellow gummy mass (Abirami et al. 2014; Madhu et al. 2014).

Acidic extraction was performed by incubating 150 g of wet weight of soft tissue in one liter of 1 N HCl for 10 min at 100 °C (Zatylny et al. 2000). After homogenization, they were centrifuged at 20,000 × g for 30 min at 4 °C. The supernatant was lyophilized to give the acidic extract (Defer et al. 2009; Abirami et al. 2014). All three crude extracts were stored at − 20 °C until antimicrobial assays were performed. In each test, all extracts were used in the same weight volume ratio.

2.3 In vitro antibacterial and antifungal assay

The antibacterial and antifungal activities of these three crude extracts of C. glaucum were determined by means of the standard agar disc diffusion method (NCCLS 1993; Bizuye et al. 2013; CLSI 2017). The extracts were tested against 16 g-negative bacteria, such as Acinetobacter (A. haemolyticus and A. lwoffii), Brevundimonas diminuta, Escherichia coli, Klebsiella (K. oxytoca and K. pneumoniae), Lelliottia amnigena, Ochrobactrum anthropi, Pseudomonas (P. alcaligenes, P. aeruginosa and P. stutzeri), Salmonella typhimurium, Shewanella algae, Shigella flexneri, Stenotrophomonas maltophilia and Vibrio alginolyticus, 7 g-positive bacterial strains, such as Bacillus (B. amyloliquefaciens, B. badius, B. pumilus and B. subtilis), Globicatella sulfidifaciens, Staphylococcus aureus and Streptococcus pyogenes, two yeast strains, such as (Candida albicans and Trichosporon asahii) and two fungal strains belong to Aspergillus (A. braselienses and A. fumigates).

One-hundred μl of each dissolved extract in dimethyl sulfoxide (DMSO) (100 μg ml−1) was loaded separately into each disc (6 mm diameter) for antimicrobial and antifungal assay, and left until dry. The loaded discs were added to the surface of cultured Petri dishes, and kept for 2 h at 4 °C to allow the diffusion of dissolved compounds into the agar. Then, the plates were incubated for 24 h at 37 °C for bacteria and 48–72 h at 28 °C for fungi. The present study used DMSO as a negative control. After incubation, the inhibition zones diameters were measured in mm using a Vernier caliper scale.

2.4 Measurement of the cytotoxicity of winter acidic crude tissue extract of Cerastoderma glaucum using 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay

This assay was performed according to Bahuguna et al. (2017). VERO (Verda reno) cells from passage number 76 were used in this assay, and grown as a monolayer in Eagle’s Minimum Essential Media (EMEM) with hanks balanced salt solution (HBSS) supplemented with 10% fetal bovine serum and 1% antibiotic solution mixture of penicillin G-sodium salt and streptomycin sulfate was added to avoid bacterial contamination (Olicard et al. 2005a, b). Eight different concentrations of the acidic extract (1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.8 µg ml−1) were applied in four replicates. Fifty μl of MTT reagent were added to wells of blank, negative cell control and different concentrations of the acidic extract. After that, the plates were incubasted in a CO2 incubator at 37 °C for 4 h for the reduction of MTT into formazan by the mitochondrial dehydrogenase activity of viable cells. One-hundred μl of dimethyl sulfoxide (DMSO) was added to each well and incubated in a CO2 incubator at 37 °C for 30 min to solubilize the purple needle crystals of formazan. Finally, the absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay (ELISA) microplate reader to determine the cellular viability. The percentage of cell survival rate was calculated by the following equation, and the maximum non-toxic concentration (MNTC) was determined.

$${\text{Survival rate }}\% = \frac{{A_{{{\text{Ex}}}} - \, A_{b} }}{{A_{c} - \, A_{b} }} \times \, 100$$

where, AEx: Absorbance of different concentrations of the acidic extract. Ab: Absorbance of the blank. Ac: Absorbance of the negative control.

$${\text{Cytotoxicity }}\% = 100 \, - {\text{Survival rate }}\%$$

2.5 In vitro antiviral activity of winter acidic crude tissue extract of Cerastoderma glaucum using MTT assay

The antiviral activity of winter acidic extract was evaluated in vitro against two viral strains; Hepatitis A virus H-10 strain and Herpes simplex virus type 1 (HSV-1, sensitive to Acyclovir that was used as a positive control of HSV-1 infection and as a reference for HSV-1 inhibition) infecting VERO cells. The determination of antiviral activity was based on the cytopathic effect inhibition assay (Langois et al. 1986). This assay was performed in eight replicates. The percentage of inhibition viral activity of winter acidic extract was calculated using the following equation:

$$\frac{{A_{{\text{virus - extract}}} {-}A_{{\text{control virus}}} }}{{A_{{\text{cellular control}}} {-} \, A_{{\text{control virus}}} }} \times \, 100$$

where Avirus-extract: absorbance of infected treated (virus-extract) well. Acontrol virus: absorbance of the control virus. Acellular control: absorbance of the cellular control.

2.6 Characterization of the different crude soft tissue extracts of Cerastoderma glaucum

2.6.1 Estimation of total protein concentration

One gram of each extract was added to water-soluble extraction buffer (a ratio of 1:2 w:v) for protein purification from the different crude tissue extracts and estimation of total protein content. The total protein concentration in all different crude soft tissue extracts of C. glaucum was determined according to Bradford (1976) method. This assay was performed in three replicates.

2.6.2 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis

SDS-PAGE was used to distinguish the total soluble protein fragments and their molecular weights distribution according to the method of Laemmli (1970). Slab gel (15%) and stacking gel (4%) were prepared. After gel polymerization, 30 µg proteins of each sample were loaded, and unstained broad range protein ladder (250–5 kDa) (Thermo Fisher, 26,630) was applied as a molecular marker. Electrophoresis was performed at 75 V through the stacking gel followed by 125 V for approximately 2 h. The gel was stained 0.1% coomassie brilliant blue R-250 for 2 h, then destained with a solution of glacial acetic acid, methanol and water (1:3:6, respectively). Gel documentation system (Gel doc-it, UVP, England) was applied for data analysis using Totallab analysis software, www.totallab.com, (Version 1.0.1). The samples were compared with the standard protein molecular weight marker (250–5 kDa).

2.6.3 Fourier transform-infrared (FT-IR) spectral analysis

One part of the lyophilized sample was mixed with 80 parts of dried potassium bromide (KBr), and compressed to prepare as a salt disc with 10 mm diameter. Then, the absorption of this disc was read using the FT-IR spectrophotometer (Germany Bruker tensor 27) between 400 and 4000 cm−1. The frequencies of different components in each sample were analyzed (Periyasamy et al. 2012; Gayathri et al. 2017).

2.7 Statistical analysis of data

One way analysis of variance (ANOVA) and post-hoc multiple-comparison (Tukey) tests were used to compare the total protein concentrations among the different crude soft tissue extracts of C. glaucum. The results were presented as means ± standard deviations (SD). Statistical analysis of data was performed using the statistical package for the social sciences IBM-SPSS software (Version 20.0). The statistical significance was set at level P ≤ 0.05.

3 Results

3.1 Antimicrobial activities of the different crude soft tissue extracts of Cerastoderma glaucum

Results shown in Table 1 indicate that winter acidic tissue extract of Cerastoderma glaucum demonstrated the highest antimicrobial activities compared to winter chloroform and methanol extracts. The maximum zone of inhibition of winter acidic tissue extract was recorded against Shigella flexneri (24 mm), and the minimum zone of inhibition was observed against Klebsiella oxytoca, Pseudomonas stutzeri, Globicatella sulfidifaciens and Bacillus (B. amyloliquefaciens, B. badius and B. pumilus) (10 mm). On the other hand, winter chloroform and methanolic extracts of C. glaucum showed activities against 4 bacterial strains with inhibition zones ranging from 12 to 13 mm and 9 to 13 mm, respectively. Furthermore, winter acidic extract of C. glaucum has antifungal activity against 2 yeast strains Candida albicans (11 mm) and Trichosporon asahii (22 mm), with no recorded activity against Aspergillus (A. fumigatus and A. braselienses).

Table 1 Inhibition zones (mm) of the three different crude soft tissue extracts of Cerastoderma glaucum collected from Lake Timsah during winter season
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Also, the very low antimicrobial activity of different tissue extracts of C. glaucum during summer season when compared with winter extracts. During summer season, antibacterial activities of the acidic extract (10 mm) were observed against 3 bacterial strains; Klebsiella pneumoniae, P. stutzeri and G. sulfidifaciens, while the activities of a chloroform extract (10 mm) appeared only against P. stutzeri and G. sulfidifaciens. But, no detectable antimicrobial activity of methanol extract was observed against all tested bacterial and fungal strains during summer season.

3.2 Cytotoxicity and antiviral activity of winter acidic extract of Cerastoderma glaucum

The winter acidic tissue extract (62.5 µg ml−1) of C. glaucum presented cytotoxicity about 24.030%. This concentration (62.5 µg ml−1) was considered to be the MNTC used in the antiviral assay of winter acidic extract of C. glaucum (Table 2). At the antiviral level, inhibition viral activities of this extract against Hepatitis A virus H-10 strain and HSV-1 virus are 62.383% and 57.035%, respectively. Also, 1.25 µg ml–1 of acyclovir which is the positive control for HSV-1 showed a low percentage of cell destruction (5.147%) (Table 3).

Table 2 Cytotoxicity of winter acidic extract of Cerastoderma glaucum at different concentrations and determination of the maximum non-toxic concentration (MNTC)
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Table 3 Antiviral activities and cytotoxicity at the maximum non-toxic concentration (MNTC) of winter acidic extract of Cerastoderma glaucum and acyclovir against viral strains
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3.3 Characterization of the different crude soft tissue extracts of Cerastoderma glaucum

3.3.1 Quantification of protein

The present results in Fig. 2 indicate that all winter extracts of C. glaucum exhibited high significance in the total protein contents compared with summer extracts (P = 0.000). Moreover, the total protein content increased significantly in winter acidic extract of C. glaucum when compared with the other extracts (P ≤ 0.01). Also, the acidic extract of C. glaucum exhibited the highest total protein contents during winter and summer seasons (9.8 and 6.3 mg ml−1, respectively) compare with chloroform and methanolic extracts.

Fig. 2
figure 2

Total protein concentrations (mg ml−1) of different crude soft tissue extracts of Cerastoderma glaucum collected during winter and summer seasons. Values are means ± standard deviation (SD). Different letters (a, b, c, d and e) are significant, *Significant at P value ≤ 0.05, **Significant at P value ≤ 0.01 and ***Significant at P value ≤ 0.001 (one-way ANOVA)

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3.3.2 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis

The electrophoretic profile of SDS-PAGE of different crude tissue extracts of C. glaucum showed active fractions of low molecular weights of peptides less than 10 kDa (Fig. 3). Four clear bands were detected in the gel of winter acidic tissue extract which represented molecular weights of proteins of 8.588, 7.237, 4.423 and 2.692 kDa. While, winter chloroform and methanol soft tissue extracts showed 3 bands with molecular weights less than 10 kDa (7.237, 4.615 and 3.654 kDa) and (7.237, 4.423 and 3.654 kDa), respectively. Also, there were 3 active peptides with molecular masses of 8.401, 6.231 and 3.654 kDa in summer chloroform extract, while only two clear bands were observed along the gel in summer methanol and acidic tissue extracts at (6.231 and 3.654 kDa) and (8.401 and 6.231 kDa), respectively.

Fig. 3
figure 3

An electrophoretic profile of 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) showing the computerized detection of protein patterns in the different crude soft tissue extracts of Cerastoderma glaucum collected during winter and summer seasons. The gel was stained with coomassie brilliant blue stain. Where, Lane 1: corresponds to the position of marker molecular masses ranging from 250 to 5 kDa; Lanes 2, 3 and 4: are different winter extracts; Lanes 5, 6 and 7: are different summer extracts

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Also, the results indicated that the protein contents of the bands with molecular masses; 7.237 and 4.423 kDa were high in winter acidic extract when compared with winter chloroform and methanol extracts. Moreover, there were two bands of molecular weights; 8.588 and 2.692 kDa in winter acidic extract, not found in the other extracts, containing protein content 1.69% and 1.56%, respectively.

3.3.3 Fourier transform-infrared (FT-IR) spectral analysis

FT-IR characterization of all crude tissue extracts of C. glaucum showed the presence of functional groups of protein like HN–C=O, ketone C=O (Amide V) and C–O stretching vibration in carboxylic group (COOH), C–N stretching vibration in primary and secondary amines, C-S linkage and C–H stretching vibration in the methylene group at appropriate wavelengths (Fig. 4). Winter acidic extract of C. glaucum had a large number of functional groups (12 groups) compare with the other extracts. All extracts, except winter and summer chloroform extracts, had peaks at 1486–1546 cm−1 which are attributed to N–H bending vibration coupled with the C–N stretching vibration (Amide II) group. Also, the S–S linkage stretching vibrations were recorded in all extracts, except summer methanol extract, at wavenumbers ranging from 529 to 598 cm−1. Furthermore, the additional bands were observed in winter acidic extract in 1651–1737 cm−1, 3385 cm−1 and 1193–1205 cm−1 peaks indicating the presence of C=O stretching vibration in COOH (Amide I), N–H stretching vibration in aliphatic primary amine and C–N stretching vibration in aliphatic tertiary amine, respectively (Table 4).

Fig. 4
figure 4

Fourier transform-infrared (FT-IR) spectral analysis of the different crude tissue extracts of Cerastoderma glaucum collected during winter and summer seasons: a Winter chloroform extract; b Winter methanol extract; c Winter acidic extract; d Summer chloroform extract; e Summer methanol extract; f Summer acidic extract

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Table 4 Functional groups of the different crude tissue extracts of Cerastoderma glaucum collected during winter and summer seasons
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4 Discussion

The marine bivalves Cerastoderma glaucum are greatly dispersed in Lake Timsah, Ismailia, Egypt. The purpose of using different solvents in preparing a tissue extract is to provide inclusive information on the properties of the bioactive substances and their activity (Fernández et al. 1996).

The present study shows high antimicrobial activity of different winter extracts compared with summer extracts. This may be due to high significance in total protein content in all winter crude extracts compare with summer crude extracts Gayathri et al. (2017). indicated that bivalve extracts are usually complex mixtures of bioactive molecules, mainly proteins and peptides which are responsible for antimicrobial and antiviral activities. Also, Sharma et al. (2009), Estari et al. (2011) and Dhanalakshmi and Sanjeevi (2016) returned the low antimicrobial activity during summer to illness stress, hypoxia, increasing water temperature and salinity and gametogenesis process during summer season. Moreover, free ions produced by the high salt concentrations in the surrounding medium, typical of some diseases, could effectively decrease the electrostatic interactions of AMPs with the negatively-charged surface of bacterial membrane and thus antimicrobial activity (Jenssen et al. 2006; Zannella et al. 2017).

The acidic extract of C. glaucum demonstrated the highest antimicrobial activities. These results are confirmed by Abirami et al. (2014) who found that the acidic extract of Meretrix casta exhibited prominent antibacterial activity. The differences in the antimicrobial activity of bivalve extracts may depend on the extraction method, extracting capacity of solvents, compound extracted and bacteria and bivalve species (Dhanalakshmi and Sanjeevi 2016; Ghorbanalizadeh et al. 2018).

The antimicrobial activity of winter acidic extract of C. glaucum ranged from 10 to 24 mm. This extract had inhibition zones against Escherichia coli, Salmonella typhimurium, Shigella flexneri and Streptococcus pyogenes. These observations agree with Arputha et al. (2013), Sugesh and Mayavu (2013) and Bibiana et al. (2014). Arputha et al. (2013) recorded the inhibitory effect of 9–12 mm, 8–13 mm, 8–16 mm, 10–23 mm and 16–26 mm with 5% acetic acid crude extracts of Donax cuneatus phenotypes (P1, P2, P3, P4 and P5, respectively). Also, the maximum inhibition zones of the acetic acid extract of D. cuneatus phenotype (P5) were observed against Streptococcus (18 mm), Salmonella (17 mm) and Shigella (17 mm) Sugesh and Mayavu (2013). recorded that the acetic acid extract of M. casta showed a 13 mm inhibition zone against E. coli. Bibiana et al. (2014) reported that the maximum inhibitory effects against S. flexneri and S. typhimurium were 25 and 19 mm, respectively in case of 5% acetic acid crude extract of D. cuneatus, and 21 and 17 mm, respectively in case of Pitar erycina extract.

Furthermore, the present study indicated that winter acidic extract of C. glaucum had antifungal activity against 2 yeast strains; Candida albicans (11 mm) and Trichosporon asahii (22 mm), with no recorded activity against Aspergillus (A. fumigatus and A. braselienses). Similar results were reported against C. albicans in the previous studies, such as Sharma et al. (2009) and Sugesh and Mayavu (2013) supporting the present antifungal results of winter acidic extract of C. glaucum. Sharma et al. (2009) found that the acid-enzyme hydrolysis aqueous (AEH-Aq) extract of Villorita cyprinoides had antifungal activity against C. albicans (13 mm) Sugesh and Mayavu (2013). reported that the methanolic, ethanolic and acetic acid extracts of M. meretrix had inhibition zones with 5, 7 and 9 mm, respectively against C. albicans.

On studying the effect of winter acidic extract against viruses, the obtained results show that 62.5 µg ml−1 concentration caused inhibition of viral activities about 62.383% and 57.035% against Hepatitis A virus and HSV-1 virus, respectively with low cytotoxicity (24.030%). These results are in accordance with Defer et al. (2009) who found that the antiviral activities of 80% solid-phase extraction (SPE)-fractions of the acidic extract from Cerastoderma edule (85 µg ml−1), Ruditapes philippinarum (125 µg ml−1), Ostrea edulis digestive gland (100 µg ml−1) and O. edulis (gills + mantle) (1280 µg ml−1) were 59.3% (24.3% of cytotoxicity), 48.7% (22.8% of cytotoxicity), 40% (34% of cytotoxicity) and 28% (20% of cytotoxicity), respectively against HSV-1.

The current study showed that winter extracts of C. glaucum exhibited high significance in the total protein contents, compared with summer crude tissue extracts. Moreover, the total protein content increased significantly in winter acidic extract of C. glaucum when compared with the other extracts. These results are confirmed with Abirami et al. (2014) who reported that methanol and acidic extracts of M. casta exhibited high amounts of protein; 786 and 745 µg mg−1, respectively, whereas the hexane and chloroform extracts exhibited low protein content of 304 and 147 µg mg−1, respectively.

From the present SDS gel electrophoresis results, all crude tissue extracts showed active fractions of low molecular weights of peptides, less than 10 kDa, representing AMPs (Hancock and Scott 2000; Mercado et al. 2005). Winter acidic extract had a higher number of low molecular weight bands compare with the other extracts. It presented four clear bands of proteins exhibited molecular weights of 8.588, 7.237, 4.423 and 2.692 kDa. Also, the results indicated that the protein contents of the bands with molecular masses; 7.237 and 4.423 kDa were high in winter acidic extract when compared with winter chloroform and methanol extracts. Moreover, there were two bands of molecular weights; 8.588 and 2.692 kDa in winter acidic extract, not found in the other extracts, containing protein contents; 1.69% and 1.56%, respectively. Chandran et al. (2009) observed only one clear band of molecular weight 9.7 kDa in the gill extraction of P. viridis using SDS gel electrophoresis, which represented a low molecular weight peptide. Also, Arputha et al. (2013) observed the crude extract of 5% acetic acid of D. cuneatus phenotype P5 to SDS-PAGE analysis, and showed bands at molecular weights of 3 and 6 kDa.

The low molecular weight peptides that were observed in C. glaucum tissue extracts, such as 6.231, 4.423, 4.615 and 3.654 kDa are very close to the molecular weights of AMPs, such as mytimycin (6.2335 kDa), myticin-A (4.438 kDa) or defensin-B (4.3924 kDa) or MGD-1 (4.418 kDa), myticin-B (4.562 kDa) and mytilin-A (3.7737 kDa), respectively. All of these AMPs are cysteine-rich polypeptides with intramolecular disulfide bonds isolated from the plasma and haemocyte granules of mussels Mytilus (M. galloprovincialis and M. edulis) (Charlet et al. 1996; Hubert et al. 1996; Mitta et al. 1999a, b, 2000a). Also, defensins have been described in oysters, such as Crassostrea gigas and C. virginica (Seo et al. 2005; Gueguen et al. 2006; González et al. 2007) and abalone Haliotis discusdiscus (De Zoysa et al. 2010). While, mytilins have been described in clam Ruditapes decussatus (Gestal et al. 2007). These AMPs display antibacterial activity against Gram-negative bacteria and Gram-positive bacteria. Mytimycin is active against Micrococcus luteus (Epand and Vogel 1999). Also, myticins A and B exhibit activity against Micrococcus (M. lysodeikticus, M. luteus), Bacillus megaterium and Enterococcus viridans, whereas only myticin-B is active against the Gram-negative bacteria, such as E. coli D31, P. aeruginosa and S. typhimurium (Mitta et al. 1999a, b, 2000b; Tincu and Taylor 2004). Moreover, Charlet et al. (1996), Romestand et al. (2003) and Gueguen et al. (2006) showed that defensins A and B had high inhibitory growth activity against the Gram-positive strains M. lysodeikticus and M. luteus than the Gram-negative strain E. coli. Furthermore, native MGD-1 has biological activity against both Gram-positive bacteria, such as B. Megaterium, M. lysodeikticus and Staphylococcus (S. aureus and S. epidermidis) and Gram-negative bacteria, such as E. coli 363, Vibrio P1, Vibrio (V. alginolyticus, V. splendidus, V. Metschnikowii) and Salmonella newport (Hubert et al. 1996; Yang et al. 2000; Romestand et al. 2003; Li et al. 2011). Mytilin-A shows considerable activity against both Gram-positive bacteria (Aerococcus viridans, Enterococcus faecalis, M. lysodeikticus, M. luteus, S. aureus and B. megaterium) and Gram-negative bacteria (E. coli, V. splendidus, V. anguillarum and S. typhimurium) (Charlet et al. 1996). Mytimycin was reported as antifungal, inhibiting the growth of Fusarium culmorum and Neurospora crassa (Charlet et al. 1996). Myticin-B and native MGD-1 are active against the fungus Fusarium oxysporum (Mitta et al. 1999a; Romestand et al. 2003). Mytilin-A showed antiviral activity on the model HSV-1 virus/VERO cells, suggesting a direct interaction of the peptide with the virus membrane (Carriel-Gomes et al. 2007).

FT-IR spectral analysis was used to detect and identify the function groups of compounds (El-Sheekh and El Kassas 2014; Abd El-Hamid et al. 2018). The present FT-IR spectral analysis revealed the occurrence of antimicrobial compound signals at different wavelengths in the crude extracts of C. glaucum. FT-IR characterization of all crude extracts of C. glaucum showed the presence of functional groups of protein like HN–C=O (Stuart et al. 1996), ketone C=O (Amide V) (Sjahfirdi and Nasikin 2012) and C-O stretching vibration (Al-Sheibany et al. 2005) in the carboxylic group (COOH), C–N stretching vibration in primary and secondary amines (Coates 2006), C–S linkage (El-Hag and Dahab 2016) and C–H stretching vibration in the methylene group (Sharma 1981; Sharma et al. 2018) at appropriate wavelengths. Winter acidic extract of C. glaucum had a large number of functional groups (12 groups) compare with the other extracts. As, there were some additional functional groups found in this extract, such as C=O stretching vibration in COOH (Amide I) (Sjahfirdi and Nasikin 2012), N–H stretching vibration in aliphatic primary amine, C–N stretching vibration in aliphatic tertiary amine (Coates 2006), N–H bending vibration coupled with C–N stretching vibration (Amide II) (Sjahfirdi and Nasikin 2012) and S–S linkage stretching vibration (Biswas et al. 2007; El-Hag and Dahab 2016) at different wavelengths.

Moreover, the present FT-IR spectral results obtained from C. glaucum tissue extracts are very close to FT-IR spectra recorded by Arputha et al. (2013) and Madhu et al. (2014). Arputha et al. (2013) noticed aldehyde C=O, amino (NH) and amine at 1707, 3396 and 1560 cm−1, respectively in the acetic acid crude extract of D. cuneatus. Also Madhu et al. (2014), observed peaks at 1654 and 1500 cm−1in the methanol tissue extract of P. viridis, which indicate the presence of the characteristic amide groups, such as C = O stretching vibration in COOH (Amide I) and N–H bending vibration coupled with C–N stretching vibration (Amide II), respectively. The bands in the range of 1520 to 1700 cm−1 are associated with HN–C=O which is the peptide group of proteins, and provides information about protein structures (Pauling and Corey 1951; Stuart et al. 1996). The bands at 1647 and 1654 cm−1, that occur at similar wavelengths in polyamides and proteins, are commonly assigned to stretching of C=O group bonded to N–H of the neighboring infra sheet chain (Focher et al. 1992).

The presence of S–S linkage and C-S linkage stretching vibrations in the different C. glaucum extracts at bands 529–598 cm−1 and 602–671 cm−1, respectively indicate the presence of cystine peptide. Baranska (2013) found that cystine peptide and cysteine amino acid containing sulfur showed an intensive band area of 500 cm−1 (S–S stretching band) as well as in the range between 2550–2580 cm−1 (SH stretching vibration band), respectively. Ameri et al. (2017) revealed that the antibacterial activity of Thais savignyi gastropod extract may be due to the presence of a disulfide (S–S) functional group (534 cm−1). Therefore, the present study suggests that the absence of antimicrobial activity of summer methanol extract is due to the absence of the S–S functional group in this extract.

5 Conclusion

In conclusion, winter acidic extract of Cerastoderma glaucum collected from Lake Timsah, Ismailia, Egypt may be a source of a large group of low molecular weight peptides, and has antimicrobial activities against both Gram-negative and Gram-positive bacteria, yeast and virus strains due to these peptides. Consequently, it could be effectively used as an alternative source of antimicrobial and antiviral drugs with subsequent health benefits. The purification and identification of these AMPs from C. glaucum will help researchers in the development of novel antibiotics.