• fatty acid;
  • growth;
  • nutrient retention;
  • Porphyra spheroplasts;
  • red sea bream


  1. Top of page
  2. Abstract

ABSTRACT:  Two test diets with and without 5% Porphyra spheroplasts (PS) were formulated using white fishmeal as the main protein source. Red sea bream Pagrus major (mean body weight 15.4 ± 0.1 g) were maintained in a flow-through system (100 L) of thermo-controlled sea water (salinity 32–34, 25°C, 8 L/min) with ordinary aeration (400–600 mL/min) under laboratory light conditions (light–dark 12 h:12 h). Fish were fed three times a day at 10:00, 14:00 and 18:00 hours by hand for 42 days at 6% body weight on each experimental diet. Studies revealed that growth performance, survival and nutrient retention were significantly (P < 0.05) higher in the groups fed a diet containing spheroplasts (PS diet). Further, the fish fed the PS diet showed significantly (P < 0.05) lower feed conversion rates. Both groups of the PS and control diets had similar levels of body nutritional profile in terms of proximate compositions and fatty acids without compromising blood serum related parameters. From these experimental results, thus, it is comprehensible that a supplementary diet containing Porphyra spheroplasts can be used for maximizing not only growth of P. major but also for utilization of the feed ingredients.


  1. Top of page
  2. Abstract

Red algae Porphyra (purple laver) is a commercially, nutritionally and culturally important species used widely in Japanese traditional foods. It is said Porphyra red algae are probably one of the healthiest sea foods on the planet.1,2 Algae contain a wide variety of complex polysaccharides in their rigid cell walls. These polysaccharides seize water and form gum-like masses in the digestive tract, increase the thickness of intestinal contents, and disturb digestion when included in fish diets. For the successful availability of all nutrients, the removal of this rigid cell wall is still considered as the most critical step. Recent innovations in marine biotechnology including the spheroplast isolation technique by using polysaccharide-degrading enzymes3,4 have shown some promise that purple laver may be used as a food additive without the cell wall. Spheroplasts are easily digestible when ingested by animals as a food ingredient. However, the cell wall of Porphyra is composed of three kinds of polysaccharides: β-1,4-mannan, β-1,3-xylan and porphyran. Therefore, the cell wall is not degraded by commercially available cellulase and pectinase, which are the enzymes that hydrolyze main polysaccharides like cellulose and pectin contained in the cell wall of terrestrial plants. In the present experiment, we prepared three enzymes (β-1,4-mannanase, β-1,3-xylanase and agarase) from bacteria isolated from natural habitats and produced spheroplasts from Porphyra by treatment with the enzymes. The dietary performance of Porphyra spheroplasts were investigated as a feed additive for raising red sea bream, which is one of the most important aquaculture target fish in Japan.


  1. Top of page
  2. Abstract

Isolation of spheroplasts

Dried thalli of Porphyra were supplied by the Saga Prefectural Ariake Fisheries Research and Development Center. The spheroplasts were prepared by digestion of Porphyra with the cell wall-degrading enzyme solution. Dried thalli of Porphyra (1 kg) were immersed in the cell wall-degrading enzyme solution, which contained three enzymes (60 g Sumizyme ACH, 200 units of agarase and 100 units of β-1,3-xylanase) in 30 L of water, and agitated at 20°C for 20 h. After centrifugation (3000 ×g, 10 min), spheroplasts (PS) were collected as the precipitate and lyophilized. Sumizyme ACH (Shin Nihon Chemical, Anjo, Japan) has β-mannanase activity. Agarase and β-1,3-xylanase were prepared from culture fluids of Vibrio sp. PO-303 and Vibrio sp. XY-214, respectively, by the method reported previously.3,4 One unit is defined as an amount of each enzyme that produces reducing sugar corresponding to the constituent monosaccharide of each substrate polysaccharide (β-1,4-mannan, agar and β-1,3-xylan). Spheroplasts were obtained and freeze-dried so the nutrient qualities were retained in good condition. Freeze-dried PS was manually smashed and ground into powder form by a mortar and pestle and incorporated into the test feed.

Test fish and experimental design

Red sea bream Pagrus major used in this experiment were reared from eggs in the laboratory. When fish were five months old, they were distributed into 100-L polycarbonate aquaria set indoors with 13 fish each (initial mean live weight 15.4 ± 0.1 g). Each aquarium was aerated (400–600 mL/min) and supplied with flow-through thermo-controlled sea water (salinity 32–34, 25°C, 8 L/min) under artificial light conditions of 12 h:12 h light–dark. Two kinds of semi-purified diets (Table 1) with and without supplementation of 5% PS (dry weight basis) were formulated using white fishmeal as the major protein source, and named the PS diet and control diet, respectively. Every morning the diets were processed into single-moist after adding 60% w/w of fresh water and fed to fish at ad libitum three times a day at 10:00, 14:00 and 18:00 hours by hand for six weeks. The daily feeding ration was approximately 6% of the mean wet body weight estimated from the growth curve obtained on periodical measurements. All the experimental fish were weighed individually every two weeks. At the end of the feeding trials, body weight and length measurements were conducted. In addition, proximate composition analysis for the dorsal muscle was carried out from pooled samples obtained from the all the test fish in each group after termination of the experiment.

Table 1.  Ingredients and proximate compositions of the experimental diets for Pagrus major
Ingredient (%)Control dietPS diet
  • Modified Halver's10 vitamin mixture +α-cellulose.

  • Modified USP XII salt mixture with trace elements (Halver10) +α-cellulose.

  • CMC, carboxymethylcellulose.

White fishmeal6562
Porphyra spheroplast05
Pollack liver oil55
Proximate composition (%) 
Crude protein51.048.7
Crude lipid9.69.4
Crude ash16.515.8

The biological parameters used for evaluating rearing results were defined and calculated as follows:

  • Specific growth rate (SGR, %/d) =  (ln Wf − ln Wi) ×  100/rearing period (day), where Wf and Wi are final and initial mean wet body weight (g), respectively.

  • Feed conversion ratio (FCR) = diet given in dry basis (g)/live body weight gain (g)

  • Protein retention rate (PRR, %) = dorsal muscle protein gain (g) × 100/protein fed (g)

  • Lipid retention rate (LRR, %) = dorsal muscle lipid gain (g) × 100/lipid fed (g)

  • Protein efficiency ratio (PER) = live body weight gain (g)/protein fed (g)

  • Condition factor (CF) = wet body weight (g) × 1000/body length3 (cm)

  • Hepatosomatic index (HSI) = wet liver weight (g) × 100/wet body weight (g)

Proximate composition and blood serum analyses

Determinations of moisture, crude protein, crude fat and ash of the diets and dorsal muscles were carried out by 10 h drying at 110°C, the semi-micro Kjeldahl method (N × 6.25), ethyl ether extraction and 5 h combustion at 600°C, respectively. Blood was withdrawn from the caudal vein by a syringe without anesthesia (2-phenoxyethanol, 500 p.p.m.). Hematocrit values were measured using a microhematocrit tube, which was sealed. All tubes were centrifuged at 15 000 ×g for 5 min. The levels of glutamic oxaloacetic transaminase (GOT, IU/L), glutamic pyruvic transaminase (GPT, IU/L), alkaline phosphatase (ALP, IU/L), glucose (mg/dL), albumin (g/dL), creatinine (mg/dL), total protein (g/dL), total cholesterol (mg/dL), triglycerides (mg/dL) and urea-N (mg/dL) were determined using an automatic analyzer (SPOTCHEM SP-4410, Arkray, Kyoto, Japan) and a commercial kit (NEFA C-test, Wako Pure Chemical, Osaka, Japan) on the blood serum of three fish from each group. The blood serum was collected as supernatant from centrifugation (4000 ×g, 5 min) of blood.

Fatty acid analysis

Total lipids were extracted using a modified procedure.5 Lipid was saponified using 2 N KOH in methanol, and fatty acids were converted to methyl esters with 5% H2SO4 in methanol. These fatty acid methyl esters were diluted in hexane and separated on a fused silica capillary column (CPB20-M25-025, Shimadzu, Kyoto, Japan) by gas–liquid chromatography (2010 GC, Shimadzu) with an FID detector. The temperature program was 150°C for 3 min, followed by an increase at a rate of 3°C/min to a final temperature of 220°C for 15 min. Peaks were identified6 by comparison with GLC standard mixtures (Supelco, Bellefonte, PA, USA).

Statistical analyses

Statistical analyses were performed using a Student's t-test and P < 0.05 was considered statistically significant.


  1. Top of page
  2. Abstract

Growth and nutrient retention

After six weeks feeding, trial fish with an initial mean body weight of 15.4 g reached 65.9 ± 2.0 g in the PS group, but only 57.0 ± 1.3 g of body weight was recorded in the control group (Table 2). There were more than 60 percentage points of difference in percent of mean live weight gain between the PS and control groups (P < 0.01). As a result, a significantly better (P < 0.01) specific growth rate (3.47 ± 0.10%/d) was obtained in the PS group than the control group (3.11 ± 0.07%/d). As for feed utilization, the PS group showed better results in FCR, PRR, LRR and PER values than the control group. On the other hand, there appeared to be no difference in the values of CF and HSI between the PS and control groups.

Table 2.  Growth and feed utilization of Pagrus major cultured with experimental diets for 42 days
 Control dietPS diet
  • NS, not significantly different.

  • *

    P < 0.05,

  • **

    P < 0.01, Student's t-test.

  • Formulae to calculate biological parameters are given in text.

  • Values are represented as mean ± standard error obtained from triplicated groups, except for the final survival.

  • Each superscript in the same row shows statistical significance.

Initial mean body weight (g)15.4 ± 0.115.3 ± 0.2NS
Final mean body weight (g)57.0 ± 1.365.9 ± 2.0*
Live weight gain (g)41.6 ± 1.350.6 ± 2.2*
Growth (% gain in body weight)269.5 ± 10.1331.2 ± 17.5*
Specific growth rate (SGR, %/d)3.11 ± 0.073.47 ± 0.10*
Final survival rate (%, range)76.9–92.394.9–100
Feed conversion ratio (FCR)1.91 ± 0.101.52 ± 0.01**
Gross protein retention (PRR, %)21.36 ± 0.728.6 ± 0.6**
Gross lipid retention (LRR, %)10.10 ± 0.417.8 ± 1.6**
Protein efficiency ratio (PER)1.04 ± 0.041.35 ± 0.01**
Condition factor (CF)30.7 ± 0.431.0 ± 0.8NS
Hepatosomatic index (HSI)1.25 ± 0.041.34 ± 0.16NS

Biochemical compositions and blood serum analyses

There were little differences in moisture, crude protein and crude ash between the PS and control groups (P > 0.05), but crude lipid showed higher values in the PS group (Table 3). The fatty acid composition of dorsal muscle is summarized in Table 4. Fatty acid profiles for the dorsal muscle of the experimental fish were similar in both groups and no significant difference was observed in the values (P > 0.05).

Table 3.  Proximate compositions of dorsal muscle of Pagrus major cultured with experimental diets for 42 days
Composition (%)InitialControl dietPS diet
  1. NS, not significantly different (Student's t-test, P > 0.05).

  2. Values are represented as mean ± standard error obtained from triplicated groups.

Moisture79.9 ± 1.176.0 ± 0.576.0 ± 1.4NS
Crude protein15.9 ± 0.219.2 ± 0.620.0 ± 0.6NS
Crude lipid1.4 ± 0.11.7 ± 0.12.3 ± 0.4NS
Crude ash2.6 ± 0.12.0 ± 0.12.1 ± 0.1NS
Table 4.  Fatty acid composition (percentage of total) of dorsal muscle of Pagrus major fed experimental diets
Fatty acidDorsal muscle
Control dietPS diet
  • Fatty acid values (percentage of total fatty acid) expressed as a percent of the total area identified in the chromatograms. Values are represented as mean ± standard error obtained from triplicate groups.

C14:01.57 ± 0.061.78 ± 0.07
C16:019.08 ± 0.0219.70 ± 0.26
C16:1n-73.78 ± 0.103.98 ± 0.14
C16:3n-60.98 ± 0.051.05 ± 0.09
C18:06.64 ± 0.116.37 ± 0.34
C18:119.94 ± 0.3320.00 ± 0.34
C18:2n-61.58 ± 0.031.45 ± 0.03
C18:3n-60.12 ± 0.000.11 ± 0.00
C18:3n-30.30 ± 0.020.28 ± 0.01
C18:4n-30.44 ± 0.330.50 ± 0.04
C20:00.14 ± 0.010.14 ± 0.01
C20:12.90 ± 0.052.95 ± 0.07
C20:2n-60.19 ± 0.000.20 ± 0.00
C20:4n-60.93 ± 0.040.85 ± 0.02
C20:4n-30.57 ± 0.010.56 ± 0.01
C20:5n-38.61 ± 0.238.86 ± 0.17
C22:11.97 ± 0.112.28 ± 0.12
C22:4n-60.24 ± 0.010.19 ± 0.01
C22:5n-32.68 ± 0.062.56 ± 0.05
C22:6n-316.42 ± 0.6314.38 ± 0.87

Blood serum (Table 5) parameters also showed no clear trend. A higher value was observed in blood for triglycerides in the PS group although values are not significantly (P > 0.05) different because of the large variation in value. On the other hand, blood serum analysis showed no adverse effect of incorporation of PS in the diet.

Table 5.  Blood serum parameters of Pagrus major fed experimental diets for 42 days
Control dietPS diet
  1. Values are represented as mean ± standard error obtained from triplicate groups.

Hematocrit (%)40.6 ± 1.0341.0 ± 2.0
Glutamic oxaloacetic transaminase (GOT, IU/L)73.9 ± 43.851.1 ± 26.7
Glutamic pyruvic transaminase (GPT, IU/L)84.7 ± 18.572.3 ± 22.3
Alkaline phosphatase (ALP, IU/L)67.0 ± 9.872.0 ± 7.5
Glucose (mg/dL)49.2 ± 6.452.0 ± 11.4
Albumin (g/dL)1.0 ± 0.01.0 ± 0.0
Creatinine (mg/dL)0.31 ± 0.00.32 ± 0.0
Total protein (g/dL)3.6 ± 0.43.8 ± 0.3
Total cholesterol (mg/dL)190.2 ± 27.0188.9 ± 29.0
Triglycerides (mg/dL)96.0 ± 41.5132.0 ± 13.7
Urea (mg/dL)5.5 ± 0.25.9 ± 0.9


  1. Top of page
  2. Abstract

In red sea bream, a Porphyra spheroplast supplemented (PS) diet improved feed utilization efficiency and enhanced fish growth and survival, although no remarkable effect was obtained in the carcass and fatty acid compositions of the fish obtained in two treatments. Also, no effect was noted in the body proportion and size of liver, the most important organ to play an essential role in many metabolic processes of nutrients, among the fish from two dietary groups. The experimental results obtained in this feeding trial show spheroplasts have great potential as a feed supplement from the viewpoint of efficient utilization of dietary protein and lipid. Nakagawa7 found that algal supplementation in fish feed was previously reported to be effective by enhancing the immune system or activating fat accumulation/mobilization as well as improving absorption, assimilation and retention of nutrients. Small amounts of various kinds of macroalgal supplementation to diets improve fish growth and feed efficiency, mainly due to the improvement of lipid metabolism.8 Enhanced feed efficiency of P. major obtained in the present experiment when fed the PS diet may also be credited to the apparent attractiveness of the diet as a food, and because it contains vitamins, minerals and essential amino acids. Physical changes (binding ability) of the diet containing PS might also have affected the palatability and the feeding performance of the test fish, and ensured there was minimal leaching and disintegration.9

Among the blood serum parameters, fish fed the PS diet showed a tendency for high blood triglycerides, possibly from enhanced lipid retention performance in the PS-fed group. Blood serum parameters were variable and not significantly different. Currently no information is available on the effect of feeding of spheroplasts in the diets of marine finfish and more investigations are needed to confirm the mode of action for finfish growth-promoting factors that are assumed to be present in spheroplasts. Feeding trials have been previously carried out with several species of only algae (raw, dry and processed); none of these dealt with spheroplasts.

The nutritional importance of Porphyra spheroplasts in finfish diets will be deliberated. Further research will be devoted to the use of varying levels of Porphyra spheroplasts in combination with other ingredients to strengthen these findings, particularly for biodefense and immunological aspects.


  1. Top of page
  2. Abstract

This study was supported by a grant program of the Agriculture, Forestry and Fisheries Research Council in Japan (Research project for utilizing advanced technologies in Agriculture, Forestry, and Fisheries. No. 1681, 2004-2006). The author expresses gratitude to Drs M Tokuda, H Furuita and J Higano of the National Research Institute of Aquaculture for technical advice and help.


  1. Top of page
  2. Abstract
  • 1
    Druehl L. Pacific Seaweeds: A Guide to Common Seaweeds of the West Coast. Harbour Publishing, Madeira Park, BC. 2000.
  • 2
    Kuhnlein HV, Turner NJ. Traditional Plant Foods of Canadian Indigenous Peoples: Nutrition, Botany and Use. Gordon and Breach Science Publishers, Philadelphia, PA. 1991.
  • 3
    Araki T, Hayakawa M, Lu Z, Karita S, Morishita T. Purification and characterization of agarases from a marine bacterium, Vibrio sp. PO-303. J. Mar. Biotechnol. 1998; 6: 260265.
  • 4
    Araki T, Tani S, Maeda K, Hashikawa S, Nakagawa H, Morishita T. Purification and characterization of β-1,3-xylanase from a marine bacterium, Vibrio sp. XY-214. Biosci. Biotechnol. Biochem. 1999; 63: 20172019.
  • 5
    Folch J, Lees M, Sloane-Stanley GA. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497509.
  • 6
    Japan Oil Chemists' Society. In: Japan Oil Chemists' Society (ed). Standard Methods for the Analysis of Fats, Oils and Related Material. Japan Oil Chemists' Society, Tokyo. 1996; 112.
  • 7
    Nakagawa H. Effective utilization of macroalgal waste as feed ingredients for aquaculture. In: SakaguchiM, HirataT (eds). Advanced Effective Utilization of Fish and Fisheries Products. NTS Press, Tokyo. 2005; 292300.
  • 8
    Mustafa MG, Wakamatsu S, Takeda T, Umino T, Nakagawa H. Effects of algae meal as feed additive on growth, feed efficiency, and body composition in red sea bream. Fish. Sci. 1995; 61: 2528.
  • 9
    Hashim H, Saat AM. The utilization of sea weed meals as binding agents in pelleted feeds for snakehead (Channa striatus) fry and their effects on growth. Aquaculture 1992; 108: 299308.
  • 10
    Halver JE. Water soluble vitamin requirement of Chinook salmon. J. Nutr. 1957; 62: 225243.