Comparative Analysis of Nutrient Composition of Caulerpa lentillifera from Different Regions

ZHANG Meijian , MA Yurong CHE Xinyi HUANG ZumeiCHEN Peng XIA Guanghua , , and ZHAO Meihui,

1) Engineering Research Center of Utilization of Tropical Polysaccharide Resources, Ministry of Education, Hainan University, Haikou 570228, China

2) Hainan Engineering Research Center of Aquatic Resources Efficient Utilization in South China Sea, Hainan University,Haikou 570228, China

3) Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China

4) College of Food Science and Technology, Hainan University, Haikou 570228, China

Abstract This study investigated the difference of nutrient composition in Caulerpa lentillifera collected from different regions.The nutrient compositions of C. lentillifera from China’s Hainan and Shandong provinces were determined and compared with those from Semporna (Malaysia), Sabah (Malaysia), Petchburi (Thailand), and two other species of seaweeds (Malaysia). The results showed that the polysaccharide and protein contents of C. lentillifera from Hainan (44.82% ± 0.98% and 12.5% ± 0.70%), Shandong(43.22% ± 1.42% and 14.7% ± 0.72%), Petchburi (59.27% ± 2.31% and 12.49% ± 0.30%), and Sabah (44.02% ± 2.01% and 19.38% ±1.48%) were higher than those of Eucheuma cottonii (26.49% ± 3.01% and 9.76% ± 1.33%) and Sargassum polycystum (33.49% ±1.70% and 5.40% ± 0.07%), respectively. The polyunsaturated fatty acid contents in C. lentillifera from Hainan (29.98%) and Shandong (22.11%) were higher than that in Semporna (16.76% ± 0.27%), Sabah (9.49%), and S. polycystum (20.34% ± 0.43%), but lower than that in E. cottonii (51.55% ± 0.57%). In Caulerpa lentillifera of Hainan and Shandong provinces, the essential amino acid(EAA)/total amino acid (TAA) ratios were 35.22% and 38.78%, respectively; and the EAA/ETAA ratios were 54.36% and 63.35%,respectively. The EAA composition of C. lentillifera was close to the ideal model of Food and Agriculture Organization/World Health Organization. C. lentillifera was rich in mineral elements, including calcium ((3315.85 ± 127.55) mg (100 g)-1 and (3728.35 ± 92.38)mg (100 g)-1), magnesium ((6715.74 ± 82.58) mg (100 g)-1 and (8128.59 ± 242.72) mg (100 g)-1), and trace elements, including iron((510.65 ± 5.47) mg (100 g)-1 and (1972.97 ± 183.35) mg (100 g)-1), selenium ((1.04 ± 0.08) mg (100 g)-1 and (0.83 ± 0.15) mg (100 g)-1),and zinc ((33.90 ± 0.13) mg (100 g)-1 and (11.75 ± 2.05) mg (100 g)-1). These results showed that C. lentillifera was more nutritious than S. polycystum or E. cottonii in terms of polysaccharide, protein, and fatty acid contents. Furthermore, both C. lentillifera species collected from Hainan and Shandong provinces show more basic nutrients. Therefore, C. lentillifera has important development and application prospects.

Key words Caulerpa lentillifera; nutrient composition; fatty acid; amino acid; mineral; comparative analysis

1 Introduction

Caulerpa lentillifera is a naturally edible green seaweed with a grape-like appearance, also known as sea grapes, which is mainly distributed in tropical and subtropical regions (Dawes, 1998). C. lentillifera is mainly cultivated in countries, such as Philippine, Vietnam, and Japan, and is often eaten with pickles or salad (Arporn and Chirapart, 2006; Matanjun and Muhammad, 2009). In recent years, C. lentillifera has become increasingly popular in fancy restaurants due to its high carbohydrate, mineral, vitamin, and unsaturated fatty acid contents. A few studies have been conducted on its potential bioactivity and show that C. lentillifera has potential anti-diabetic activity (Sharma and Dong, 2014; Sharma et al., 2015),can prevent hypertension (Joel et al., 2018), contains antioxidants, has anticoagulant and anticancer activity (Maeda et al., 2012; Liang et al., 2015), as well as antitumor and microbial therapeutic effects (Maeda et al., 2012). Japan is the largest producer and consumer of C. lentillifera, with an annual output of about 600 tons (Song et al., 2018). In recent years, with the successful introduction of C. lentillifera in Hainan, Shandong, Guangxi, and other provinces,C. lentillifera has received considerable attention in China, and the planting and consumption have increased annually.

However, like most plants, the nutrients of C. lentillifera are affected by external factors, such as the season,climate, sampling conditions, and the sea environment where C. lentillifera grows (Van Tang et al., 2011; Mohamed et al., 2012). A few studies have reported the chemical composition of C. lentillifera from some regions of the world, but no reports are available on the changes in nutrient composition of C. lentillifera in Hainan and Shandong Provinces of China (The following article uses the origin to represent the C. lentillifera produced in this area.), which were introduced from Vietnam or Japan. In this study, we measured the nutritional and chemical composition of C. lentillifera from Hainan and Shandong,i.e., proximate composition, and vitamin, mineral, fatty acid, and amino acid contents, and we conducted a comparative evaluation of nutritive value with C. lentillifera from other regions, and two other seaweed species. The aim of this study was to comprehensively evaluate the nutritional value of C. lentillifera in different regions and provide a basis for the development and utilization of C.lentillifera.

2 Materials and Methods

2.1 Materials and Reagents

Samples of C. lentillifera were collected from culture ponds in Hainan and Shandong Provinces in China and were identified by Prof. Fang Zaiguang of Hainan University. Algae with a normal color and full particles were selected as experimental samples. The surface epiphytes were first removed with tap water, following by three washes in deionized water, and a small amount of fresh sample was used to determine moisture content; other samples were freeze-dried and used to determine basic nutrient, amino acid, mineral, and fatty acid contents.

All chemicals used in this research were of analytical grade and were purchased from chemical companies.Phenol, anhydrous copper sulfate, and thiourea were obtained from Xilong Science Co., Ltd. (Guangdong, China).Concentrated sulfuric acid, potassium hydroxide, absolute ethanol, ether, potassium sulfate, and boric acid were obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China). Methyl red indicator, bromocresol green indicator, and methylene blue indicator were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Thirty-seven fatty acid mixed standards, methyl pentadecanate, a vitamin C standard, and a vitamin E standard (both chromatographically pure) were obtained from Sigma Aldrich Co. (Shanghai, China).

2.2 Analytical Methods

2.2.1 Proximate analysis

The moisture content of the dried samples was determined gravimetrically at 105℃ until constant weight of the sample was achieved (typically 2 h) using an HB43-S Halogen Moisture Analyzer (METTLER TOLEDO International Trading Co., Ltd., Shanghai, China) (AOAC,934.01). Ash was determined after combusting the dried sample at 550 ± 25℃ for 4 h in a laboratory muffle furnace (AOAC, 930.05). Crude protein of the sample was estimated by the Kjeldahl method (Jinan Haineng Instrument Co., Ltd., K9840) (N x 6.25; AOAC, 981.10) (Liu et al., 2018). Crude fat of the sample was extracted according to Soxhlet extraction with petroleum ether (AOAC,991.36). Crude fiber was successively hydrolyzed by boiling 1.25% H2SO4and 1.25% KOH for 30 min, respectively. Total dietary fiber and insoluble dietary fiber were determined by an enzyme-gravimetric method (AOAC,991.43). Crude polysaccharide content was determined by the sulfuric acid-phenol method, and glucose was used as a standard for colorimetric determination.

2.2.2 Vitamin contents

The vitamin E in the sample was determined by a high performance liquid chromatography system (Shimadzu Co.,Tokyo, Japan) equipped with a C30column and column temperature of 20℃. The mobile phases were water (A)and methanol (B). The UV detection wavelength for vitamin E is 294 nm (GB 5009.82). Vitamin C content was determined with 2,4-dinitrophenylhydrazine.

2.2.3 Fatty acid composition

The composition analysis of 35 fatty acids in the sample was carried out by a Trace 1310 ISQ gas chromatography (GC) mass spectrometry (MS) system (Thermo Fisher Scientific Co., Waltham, MA, USA) equipped with a capillary column (30 m × 0.25 mm × 0.25 μm) and flame ionization detector (FID). The GC analysis conditions were as follows: injection temperature was 290℃, carrier gas flow rate was 1.2 mL min-1with splitless injection,and the valve opening time was 1 min. The temperature was maintained at 80℃ for 1 min. Then the temperature was increased to 200℃ at a rate of 10℃min-1, and was further increased to 250℃ at a rate of 5℃min-1. Finally it was increased to 270℃ at a rate of 2℃min-1for 3 min.The MS analysis conditions were ion source and transmission line temperature of 280℃, and the ion source was an EI source of 70 eV. Solvent delay time was 5.00 min, and the detection scan range was 30–400 amu.

2.2.4 Amino acid composition

The amino acid composition analysis of the samples was carried out using an amino acid analyzer (Hitachi High-Tech, Tokyo, Japan; L-8900) equipped with a sulfonic acid type cationic resin column (GB 5009.124). The sample was first hydrolyzed with acid, filtered through a 0.22 μm filter, and transferred to an instrument vial for use. The mixed amino acid standard working solution and the sample measuring solution were respectively injected into the amino acid analyzer in the same volume, and the concentration of the amino acid in the sample was calculated by the peak area using an external standard (Liu et al.,2011).

2.2.5 Mineral contents

Contents of macroelement including potassium (K), calcium (Ca), sodium (Na), and magnesium (Mg) were determined by inductively coupled plasma spectrometry (Thermo Fisher Scientific; ICE3500). Contents of trace element contents including zinc (Zn), iron (Fe), copper (Cu),manganese (Mn), lead (Pb), chromium (Cr), selenium(Se), and iodine (I) were also determined by inductively coupled plasma mass spectrometry (Agilent Technologies,Palo Alto, CA, USA; 7900 ICP-MS) (GB 5009.268).

2.3 Statistical Analysis

All results are expressed as mean ± standard deviation(n = 3). Significance differences were at P < 0.05, and were detected by one-way analysis of variance followed by Duncan’s multiple range test using SPSS system version 17.0 for Windows (SPSS Inc., Chicago, IL, USA).

3 Results and Discussion

3.1 Proximate Analysis

This experiment determined the basic nutrients of C.lentillifera collected from Hainan and Shandong provinces. The stalks of C. lentillifera were crystal clear and full of water. The approximate composition based on the dry weight of the analyzed samples is shown in Table 1.The seaweeds from various regions were rich in polysaccharides. The polysaccharide content in plants from Petchburi (59.27%) (Ratana-Arporn, 2006) was higher than those from Hainan (44.82% ± 0.98%), Shandong (43.22% ± 1.42%),and Sabah (44.02% ± 2.01%) (Nagappan and Vairappan,2014), whereas Sempoma polysaccharide content was the lowest (38.66% ± 0.96%) (Matanjun and Muhammad, 2009).The polysaccharide content of C. lentillifera in all of the regions (except Sempoma) was higher than those of E.cottonii (26.49% ± 3.01%) and S. polycystum (33.49% ±1.70%) (Matanjun and Muhammad, 2009). A difference was observed in the polysaccharide content among the five different geographical species of C. lentillifera. This difference may be due to the different geographical densities of each population, which affects photosynthesis, resulting in the accumulation of photosynthetic products,i.e., different polysaccharide contents. Studies have shown that the polysaccharide extract of C. lentillifera promotes immune-stimulating activity (Sun et al., 2017), has antioxidation properties, and protects against type-2 diabetes mellitus (Sharma and Dong, 2014).

Table 1 Approximate compositions (% dry weight) of Caulerpa lentillifera from different regions

In addition to the Sabah protein content, which was as high as 19.38% ± 1.48%, the protein contents in Hainan(12.5% ± 0.70%), Shandong (14.7% ± 0.72%), Sempoma(10.41% ± 0.26%), and Petchburi (12.49% ± 0.3%) plants were not significantly different. The protein contents of S.polycystum (5.40% ± 0.07%) and E. cottonii (9.76% ± 1.33%)were the lowest compared to all of the C. lentillifera samples. It has been reported that algal proteins have anticancer activity (Li et al., 2011; Reddy et al., 2003), antiinflammatory (Cherng et al., 2007), anti-oxidation and anti-radiation effects (Lu et al., 2006; Zhou, 2003). They are highly regarded by the food, bio-pharmaceutical, and medical industries.

Seaweed from Hainan (12.98% ± 1.59%) had the most abundant crude fiber content. Plants from Shandong (8.87%± 0.74%) had a fiber content which is lower than that from Hainan, but higher than those from Sempoma (1.91% ±0.0%), Sabah (4.12% ± 0.16%), and Petchburi (3.17% ±0.21%). The total dietary fiber of S. polycystum (39.67%± 0.56%) was slightly higher than all of the C. lentillifera samples, whereas the soluble dietary fiber of S. polycystum (5.57% ± 0.28%) was the lowest. Algal dietary fiber helps reduce the risk of chronic diseases, such as diabetes,heart disease, and cancer (Eyre et al., 2004), and C. lentillifera is a good source of dietary fiber. The ash content of the five species of C. lentillifera, i.e., 24.21%–37.15%,was considerably high; the ash content of S. polycystum(42.40% ± 0.41%) and E. cottonii (46.19% ± 0.42%) was higher than those of all of the C. lentillifera samples, and the difference may be related to the salinity of seawater.

C. lentillifera contained a small amount of fat. Samples from Hainan had a fat content (2.32% ± 0.23%) which was lower than that from Sabah (2.87% ± 0.03%), but slightly higher than those from Shandong (1.91% ± 0.05%),Sempoma (1.11% ± 0.05%), and E. cottonii (1.1% ± 0.05%),and much higher than those from Petchburi (0.86% ± 0.10%)and S. polycystum (0.29% ± 0.01%). In the analysis of fatty acid composition, the C. lentillifera samples were rich in unsaturated fatty acids. C. lentillifera is also rich in vitamin E, which has an anti-oxidative effect, improves blood circulation, regulates fertility (Sun et al., 2017), and helps to inhibit LDL oxidation and thromboxane formation (Matanjun et al., 2010).

3.2 Fatty Acid Composition

The total lipid contents in C. lentillifera collected from different regions were within the range of 0.86%–2.87%.Data on fatty acid composition of C. lentillifera from Hainan and Shandong are presented in Table 2. Fatty acids contents were ranged from C8 to C24, and included 15 types of saturated fatty acids, 9 types of monounsaturated fatty acids, and 11 types of polyunsaturated fatty acids (PUFAs). C. lentillifera was rich in unsaturated fatty acids. The unsaturated fatty acids in samples from Hainan and Shandong accounted for 44.35% and 36.34% of the total fatty acids, respectively, of which PUFAs accounted for 29.98% and 22.11%, respectively. The contents of unsaturated fatty acids in C. lentillifera from Sempoma was 53.59%, and the content of polyunsaturated fatty acids(16.76%) was lower than those of samples from Hainan and Shandong, but the content of monounsaturated fatty acids was higher (36.83%). Unsaturated fatty acid content in E. cottonii (74.83%) was higher than those of C. lentillifera and S. polycystum (48.70%). Only some fatty acids in samples from Sabah and Petchburi were tested. Variations in fatty acid contents were due to both the environmental and genetic differences mentioned above.

Linoleic acid and linolenic acid are two essential fatty acids that cannot be synthesized in the human body. The contents of linoleic acid in samples from Hainan (10.89%)and Shandong (10.99%) were slightly higher than those from Sempoma (7.64%) and Petchburi (4.26%). Linoleic acid helps to lower serum cholesterol and has antiatherosclerosis activities, and a lack in the diet (salt form of the acid) can cause mild skin and hair loss (Cunnane and Anderson, 1997). The linolenic acid content in samples from Hainan was 13.89%, which was higher than those from Shandong (8.26%) and Sempoma (5.85%). In the human body, linolenic acid is converted into docosahexaenoic acid and eicosapentaenoic acid (EPA), and shows a blood pressure-lowering effect (Ogawa et al., 2009). In addition, C. lentillifera also contains a small amount of EPA, and the contents of EPA in samples from Hainan,Shandong, and Sempoma were 0.39%, 0.26%, and 0.83%,respectively.

3.3 Amino Acid Composition

The amino acid composition of C. lentillifera from Hainan and Shandong revealed 16 types of amino acids with reasonably resolved separations, including 7 types of essential amino acids (EAAs) and 9 types of non-essential amino acids (NEAA) (Table 3). All of the EAA were detected in the samples in different proportions except for tryptophan. Probably the small amount of tryptophan was destroyed during the hydrolysis process. EAA content was the highest in samples from Shandong (57.01 mg g-1),followed by the samples from Petchburi (47 mg g-1), Sempoma (48.98 mg g-1), and Hainan (44.02 mg g-1). The EAA contents of S. polycystum and E. cottonii were 47.13 and 32.07 mg g-1, respectively. The protein quality and quantity are important when assessing the nutritional value of a food product. According to the ideal model recommended by the FAO/WHO, protein with better quality has an EAA/TAA ratio of 35.38% and an EAA/NEAA ratio >60% (FAO/WHO, 1973). The EAA/TAA ratios in samples from Shandong and Semporna were 38.78% and 48.19%, respectively, and the EAA/NEAA ratios were 63.35% and 90.57%, respectively, which fully met the ideal model recommended by FAO/WHO.

Glutamate (Hainan 13.82 mg g-1and Shandong 14.72 mg g-1), glycine (Hainan 19.23 mg g-1and Shandong 18.27 mg g-1), and aspartic acid (Hainan 12.47 mg g-1and Shandong 14.89 mg g-1) accounted for a large proportion of the dry samples. Glutamate and aspartic acid are umami amino acids, and glycine is a sweet amino acid. Therefore, C.lentillifera has a rich seaweed flavor that can be used as a seafood flavoring and processed into various flavored snacks.

Table 3 Amino acid content (g (100 g)-1 sample dry basis) of Caulerpa lentillifera from different regions

3.4 Mineral Contents

The ash contents of C. lentillifera collected from different locations were at high levels, meaning the high quantities of mineral elements. The contents of macroelements (Ca, K, Na, and Mg,) and trace elements (Mn,Fe, Cu, Se, Zn, and I) are shown in Table 4. Samples from Hainan (3315.85 ± 127.55 mg (100 g)-1) and Shandong(3728.35 ± 92.38 mg (100 g)-1) had the highest calcium content, followed by those from Sempoma (1874.74 ±0.20 mg (100 g)-1) and Petchburi (780.00 mg (100 g)-1).The calcium content of S. polycystum (3792.06 ± 0.51 mg(100 g)-1) was similar to that of C. lentillifera from Shandong, but the calcium content of E. cottonii (329.69 ± 0.33 mg (100 g)-1) was much lower. Calcium is the basic raw material for bone development and maintains the contraction of muscles and the transmission of nerve impulses.Magnesium can enhance memory and protect the heart.The Hainan samples (6715.74 ± 82.58 mg (100 g)-1) had a slightly lower magnesium content than those from Shandong (8126.59 ± 242.72 mg (100 g)-1), but were higher than those in other seaweeds. The Hainan (510.65 ± 5.47 mg (100 g)-1) and Shandong (1972.97 ± 183.35 mg (100 g)-1) samples contained a large amount of iron, whereas only trace amounts of iron were detected in the other seaweeds. Iron contributes to metabolism and promotes enzyme activity. The selenium contents in the Hainan,Shandong, and Sempoma samples were 1.04 ± 0.08, 0.83± 0.15, and 1.07 ± 0.0 mg (100 g)-1, respectively. Selenium has a synergistic anti-tumor effect by maintaining DNA stability, cell cycle arrest, and apoptosis (Zuo et al., 2012).In addition, seaweeds from the five different regions were rich in zinc and iodine, which play important roles in human growth and immune function.

Table 4 Mineral contents (mg (100 g)-1 sample dry basis) of Caulerpa lentillifera from different regions

The Na/K ratios of C. lentillifera collected from Hainan and Shandong were 2.63 and 1.78, respectively, which were lower than the algae from the other two regions. It has been reported that the Na/K ratios in red and brown algae are less than 1.5 (Rupérez, 2002). The Na/K ratios were very low in S. polycystum and E. cottonii. If the Na/K ratio is too high, it is detrimental to the sodium/potassium balance in the human body, which can result in cardiovascular disease. C. lentillifera was rich in minerals to meet the needs of the human body. A simple desalting operation, such as soaking, is recommended before eating.C. lentillifera can be used together with other foods, but attention should be paid to the Na/K ratio.

4 Conclusions

In this study, the nutrients in C. lentillifera collected from Hainan and Shandong provinces were analyzed comprehensively. They were compared each other as well as with two other species of seaweed reported previously.Due to different factors, such as the climate, geographical environment, and seawater quality of the growing region,the nutrients in the C. lentillifera samples differed. It’s found that the polysaccharide contents in samples from Hainan and Shandong were higher than those in E. cottonii and S.polycystum. The rich content of C. lentillifera polysaccharide is a potential source of plant polysaccharides. The EAA composition of samples from Shandong was more in line with the ideal FAO/WHO model. Due to the high protein levels and balanced amino acid models of C. lentillifera,they also showed potential as a good source of protein supplements. In addition, the respective unsaturated fatty acid contents in samples from Hainan and Shandong were 44.35%and 36.34%, of which the PUFA content was higher than in samples from Semporna, Sabah, and S. polycystum. The Hainan samples had a higher fiber content than those from Shandong, Sempoma, Sabah, and Petchburi. The ash content of all of the C. lentillifera samples was lower than that of S. polycystum and E. cottonii.

Although there were differences in nutrient composition between C. lentillifera collected from Hainan and Shandong provinces, this species has obvious nutritional benefits compared with S. polycystum and E. cottonii.Therefore, C. lentillifera can be developed as a functional food with good prospects.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 31860450), the Marine and Fisheries Bureau of Haikou (No. HHCL201804), the Innovative Research Projects of Graduate Students of Hainan Province (No. Hys2018-197), the Key Science and Technology Program of Haikou City (No. 2017051), and the Scientific Research Foundation of Hainan University(No. kyqd1662).