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Bioresource Technology 99 (2008) 77237729

Utilisation of Chlorella vulgaris cell biomass for the production

of enzymatic protein hydrolysates
Humberto J. Morris a,*, Angel Almarales b, Olimpia Carrillo c, Rosa C. Bermudez a
a
Center for Studies on Industrial Biotechnology (CEBI), University of Oriente, Avenida Patricio Lumumba s/n, Santiago de Cuba 5, CP 90 500, Cuba
b
Center of Technological Applications for Sustainable Development (CATEDES), Guantanamo, Cuba
c
Department of Biochemistry, Faculty of Biology, University of Havana, Vedado, Ciudad Habana 4, CP 10 400, Cuba

Received 19 July 2005; received in revised form 28 January 2008; accepted 29 January 2008
Available online 24 March 2008

Abstract

Studies on enzymatic hydrolysis of cell proteins in green microalgae Chlorella vulgaris 87/1 are described. Dierent proteases can be
used for production of hydrolysates from ethanol extracted algae. The inuence of reaction parameters on hydrolysis of extracted bio-
mass with pancreatin was considered, and the composition of hydrolysates (Cv-PH) was investigated in relation to the starting materials.
Signicant changes in the degree of hydrolysis were observed only during the rst 2 h and it remained constant throughout the process.
An enzyme-substrate ratio of 3045 units/g algae, an algae concentration of 1015% and pH values of 7.58.0 could be recommended.
Dierences in the chromatographic patterns of Cv-PH and a hot-extract from Chlorella biomass were observed. Adequate amounts of
essential amino acids (44.7%) in relation to the reference pattern of FAO for human nutrition were found, except for sulfur amino acids.
Cv-PH could be considered as a potential ingredient in the food industry.
2008 Elsevier Ltd. All rights reserved.

Keywords: Green microalgae; Chlorella; Enzymatic hydrolysis; Protein hydrolysate; Food industry

1. Introduction and food supplements (Iwamoto, 2003; Merchant et al.,

Since microalgae provide an ecient mean of converting
solar energy into biomass (Hall and Rao, 1994), biotech-
nology of microalgae has gained importance in recent years
due to the development of new production and environ-
mental technologies (Pulz et al., 2000). Because their
growth requires unexpensive substrates, microalgae can
be used as economical and eective biocatalysts to obtain
high added-value compounds and during productive pro-
cesses, the algal biomass formed may be used as a food
source such as proteins (Olaizola, 2003; Shimizu and Li,
2006).
The unicellular green algae (Chlorophyta, Chlorophy-
ceae) are suitable for protein products sold as health foods
*
Corresponding author. Tel.: +53 22 632095; fax: +53 22 632689.
E-mail address: hmorris@cebi.uo.edu.cu (H.J. Morris).
2002; Oh-Hama and Miyachi, 1988; Pulz and Gross,
2004). However, intact green algae Chlorella and Scenedes-
mus have a low protein digestibility due to their strong wall
(Shelef and Soeder, 1980). The enzymatic hydrolysis of cell
proteins has been described as a promising method to
improving algae protein digestibility, which makes the
product useable in human nutrition (Stoilov et al., 1995;
Tchorbanov and Bozhkova, 1988).
Progress in hydrolysis techniques has led to the produc-
tion of hydrolysates of many food proteins, using proteolytic
enzymes such as pancreatic proteases, bacterial proteases
and pepsin (Guadix et al., 2000; Kislukhina, 2002). Because
enzymatic protein hydrolysates appear to be more eective
than either intact protein or free amino acids, they have been
widely used in specic formulations with clinical applica-
tions (Frokjaer, 1994; Vioque et al., 2004).
The sources most commonly used in nutritional prod-
ucts are casein and whey proteins and soybean proteins

0960-8524/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.01.080
7724 H.J. Morris et al. / Bioresource Technology 99 (2008) 77237729
(Clemente et al., 1999). In this sense, green microalgae bio-
mass would represent in tropical countries an innovative
proteinaceous bioresource for developing enzymatic pro-
tein hydrolysates suitable for pharmacological nutrition
(Morris et al., 2007). An extended knowledge of the protein
quality and functional properties in microalgae hydroly-
sates would be useful in understanding their use as poten-
tial additives for food and dietary items. This paper
reports the results of a study on the enzymatic hydrolysis
of Chlorella vulgaris biomass with pancreatin and also,
the biochemical analysis of the obtained hydrolysates.
2. Methods
2.1. Microorganism, cultivation conditions and biomass
processing
Algae samples were obtained by autotrophic outdoor
cultivation of C. vulgaris 87/1 in open circulating cascade
systems of 500 m2 from September to December. This
strain was isolated from Chalons dam in Santiago de Cuba
and is deposited at the culture collection of the Solar
Energy Research Center (CIES). The growth medium con-
tained (g L 1): NH4NO3 (1.2), MgSO4
7H2O (1.0) and a
food grade NPK (8:12:12) fertilizer formula (0.9). The algal
suspension was bubbled with 1% CO2. The algae were har-
vested by continuous-ow centrifugation (separator Alfa
Laval, Sweden) up to 10% dry matter in the slurry and then
submitted to disruption in a DynoMill KDL apparatus
with a disruption time of 3 min. The dark-green algae
slurry was spray dried in a Niro Atomizer drier (input
200210 C, output 8090 C). The powder thus obtained
(moisture content 7%) was preserved in plastic boxes for
further use. Dry algae samples of 500 g were extracted with
ethanol (2L) at 45 C for 3 h via gentle agitation.
2.2. Chemical analysis
Total nitrogen, total bre and ash were analyzed accord-
ing to AOAC (1995) approved methods. Crude protein
content was calculated using a conversion factor of 6.25.
Total soluble sugars were estimated colorimetrically by
the phenolsulfuric acid method using a standard curve
of glucose (Dubois et al., 1956), and total lipids were mea-
sured according to Kochert (1978). Pigments were deter-
mined in methanolic extracts according to Wellburn
equations (1994). Nucleic acids (RNA + DNA) were mea-
sured as described by Rut (1973). In vitro protein digestibil-
ity (IVPD) was estimated measuring the pH of the protein
suspension immediately after 10 min of digestion with a
multienzyme solution of trypsinchymotrypsinpeptidase.
It was found that the pH of a protein suspension thus mea-
sured was highly correlated with the in vivo apparent
digestibility of rats (Hsu et al., 1977). The enzymes, trypsin
(porcine pancreatic trypsin type IX), chymotrypsin (bovine
pancreatic chymotrypsin type II) and peptidase (porcine
intestinal peptidase grade III) were purchased from Sigma
Chemical Co. (St. Louis, MO, USA).
The amino acid prole was determined by reverse phase-
HPLC after 24 h hydrolysis at 110 C with 6 mol/L HCl
and further derivatisation with phenylisothiocyanate. The
tryptophan content was analyzed by derivative spectropho-
tometry (Fletouris et al., 1993). The amino acid scores
(essential amino acid content in test protein hydrolysate/
content of same amino acid in reference protein) were cal-
culated. The lowest value corresponding to the limiting
amino acid was designated as the chemical score. The
FAO amino acid requirement pattern for the 25 year
old child was used as the reference protein for comparison.
This pattern is used, because it is the most demanding of
any age group other than infants (FAO/WHO/UNU,
1985).
In protein hydrolysates, free amino acids content was
estimated by the colorimetric ninhydrin reaction (Kalant,
1956) and soluble hydrolysed protein was assessed by the
Biurets method according to Lu et al. (2005).
All the chemicals used were of analytical grade.
2.3. Enzymatic hydrolysis
The inuence of the nature of proteolytic enzymes on the
hydrolysis of Chlorella cell proteins in ethanol extracted
biomass was studied using the following proteases: pancre-
atin, pepsin and papain (MERCK), trypsin (Leciva, Praha),
bromelain (UNICA, Cuba) and a crude extract obtained by
submerged culture of Bacillus subtilis CEBI Bs-06-4. The
specic activities and optimum pH for these proteases are
showed in Table 1. One proteolytic unit was expressed as
the amount of enzyme necessary to catalyze at an initial rate
the release of 1 lmol tyrosine from a 2% denatured casein
solution at 37 C and the optimum pH for each enzyme
within 1 min (Anson, 1938). The hydrolysis was carried
out for 4 h at 20 U/g, algae concentration of 10%, temper-
ature of 37 C and the pH considered as optimum. The
enzyme reaction was stopped by heat treatment at 85 C
for 15 min. The slurry thus obtained was centrifuged at
3000g for 10 min and the supernatants were used further
for analysis.
Table 1
Specic activity (U/mg of protein) and optimum pH of proteases used on
the enzymatic hydrolysis of cell proteins of Chlorella vulgaris 87/1
Proteolytic enzymes Specic activity Optimum pH
(U/mg of protein)a
Pancreatin 0.470 7.5
Pepsin 3.280 2.0
Trypsin 0.070 7.5
Papain 0.130 7.0
Bromelain 1.197 7.0
Crude of Bacillus subtilis 0.030 8.5
a
One proteolytic unit was expressed as the amount of enzyme necessary
to catalyze at an initial rate the release of 1 lmol tyrosine from a 2%
denatured casein solution at 37 C and the optimum pH for each enzyme
within 1 min.
 H.J. Morris et al. / Bioresource Technology 99 (2008) 77237729 7725

The potential main eects of dierent factors in the were obtained at the 5% level, dierences between individ-
hydrolysis of Chlorella proteins with pancreatin were fur- ual means were tested using the StudentNewmanKeuls
ther investigated. An algal suspension in water was hydro- test. To assay the changes in the degree of hydrolysis with
lysed at 50 C for 4 h in a 1000 mL reaction vessel, pancreatin of ethanol extracted and non-extracted algae,
equipped with a stirrer, thermometer and pH electrode. the MannWhitney test was used.
During hydrolysis of Chlorella proteins, pancreatin was
active up to 4550 C, presumably due to the protective
3. Results and discussion
eects exerted by the presence of a high concentration of
other proteins in pancreatin, and carbohydrates in cell bio-
The high protein content of certain microalgae was one
mass, as well as the conduction of hydrolysis in conditions
of the reasons to select these organisms as unconventional
where substrate saturation was ensured. The eects of algae
protein sources. Most of the cultivated microalgae have a
extraction with ethanol, the initial enzyme concentration
relative thick cellulosic cell wall which makes the untreated
[E0], the algal slurry concentration and pH were investi-
algae practically indigestible to monogastric animals or
gated by varying each factor one at a time, keeping the rest
humans (Becker, 1994). Chemical composition and in vitro
constant. Data shown in the gures and tables specify the
protein digestibility of C. vulgaris 87/1 biomass and the
conditions in which experiments were carried out. Aliquots
pancreatin hydrolysate are shown in Table 2. The main fea-
were taken out during the hydrolysis and the enzyme reac-
ture was their high protein content (P45%). These values
tion was stopped by heat treatment at 85 C for 15 min.
are comparable with those obtained in other freshwater
In all the experiments, the degree of hydrolysis (DH),
microalgal species considered as possible sources of alimen-
dened as the percentage of peptide bonds cleaved, was
tary protein (Abalde et al., 1991; Becker, 1994; Iwamoto,
measured by determination of free amino groups by the
2003).
potentiometric titration of samples to pH 9.0 with
Although the crude protein content of disrupted bio-
0.1 mol/L NaOH in presence of an excess of formaldehyde
mass of C. vulgaris 87/1 not treated with ethanol accounted
(USP 27, 2004). Each milliliter of 0.1 mol/L NaOH con-
for 50.5% of the dry matter, higher than the extracted
sumed in the titration is equivalent to 1.4 mg of a-amino
algae, the in vitro protein digestibility of biomass was of
nitrogen. Total number of amino groups was determined
70.4%. The pretreatment of the algal biomass with ethanol
by acid hydrolysis (HCl 6 mol/L) at 110 C for 24 h.
could be considered as an approach for the manipulation
of the colour of Chlorella-based products, leading to a
2.4. Gel ltration
more palatable supplement. Ethanol extracted samples
vary only marginally in total crude protein, with a higher
Samples (0.02 g) of an aqueous hot-extract from ethanol
value of IVPD (75.9%).
extracted Chlorella biomass or Cv-PH were dissolved in
Because uric acid is produced in humans and other
0.1 mol/L phosphate buer pH 6.8 and applied to a column
mammals in purine metabolism and high levels of this
of Sephadex G-100 (1  60 cm). Volume injected and sam-
metabolite may cause pathological conditions such as gout,
ple concentration were 200 lL and 2.0 mg of protein per
mL, respectively. The eluent was the above cited buer at
a ow rate of 12 mL/h. Elution was monitored at 214 nm Table 2
Chemical composition and in vitro protein digestibility (IVPD) of biomass
and the approximate molecular masses were determined
and protein hydrolysate (Cv-PH) of Chlorella vulgaris 87/1*
using the following molecular weight standards from Phar-
Constituent Non-extracted Ethanol Cv-PH
macia (Uppsala, Sweden): blue dextran (2000 kDa), bovine
biomass extracted
serum albumin (67 kDa), horse-radish peroxidase (44 kDa) biomass
and cytochrome c (12 kDa).
Crude protein (N  6.25) 50.5 0.2a 45.0 0.2b 49.7 0.3a
Gel ltration of the low molecular weight fraction of Soluble carbohydrates 17.7 0.3b 16.0 0.4b 24.0 0.4a
Cv-PH was performed in a Sephadex G-25 column. Vol- Total bre 8.5 0.5a 8.2 0.1a
ume injected and sample concentration were 200 lL and Fat 7.0 0.1a 0.3 0.09b 0.2 0.08b
2.0 mg of peptides per mL, respectively. The approximate Ash 8.3 0.3a 8.2 0.2a 8.0 0.2a
Nucleic acids 6.1 0.3a 3.8 0.4b Non-tested
masses of peptides were estimated using the following stan-
Pigments
dards: aprotinine (6.5 kDa), bovine glucagon (3.5 kDa) Chlorophylls 530.0 2.8a 25.0 3.6b
and cyanocobalamin (1.4 kDa). Carotene 220.0 0.5a 10.0 0.2b
IVPD 70.4 1.8c 75.9 1.4b 97.2 2.3a
2.5. Statistical analysis Means without the same letter are signicantly dierent at the 5% level
according to the MannWhitney test (total bre, nucleic acids and pig-
All data (arithmetic mean SE of three replicates) were ments) and the StudentNewmanKeuls test (rest of the constituents).
(): non-detectable in Cv-PH.
analyzed using the Statistical Package for Social Sciences *
Data represent the mean SE of three independent determinations.
(SPSS) version 12.0/2003 for Windows (SPSS Inc., 1989 The constituents are expressed as g 100 g 1 of dry matter, with the
2003). One way ANOVA was carried out to compare the exception of pigments (mg 100 g 1). Total bre and pigments were not
means of dierent treatments; where signicant F values detectable in Cv-PH.
7726
there is a constant worry in the utilisation of microbial cells
as food or feed due to their high nucleic acid content
(Anupama, 2000). The reduction observed in nucleic acid
concentrations in the ethanol extracted biomass is still
another very important advantage of the proposed method.
The ethanol extracts take up 1215% of the algal biomass
and contain a number of biologically active compounds as
chlorophylls, carotenoids, sterols and polyunsaturated fatty
acids. The low toxicity and the low cost were other impor-
tant reasons in choosing ethanol for algae extraction.
All known approaches to improving algae protein
digestibility for special foods aord only protein hydroly-
sates. Various physical and chemical means are known
for the treatment of algal biomass and enhancing protein
hydrolysis, including the extraction of lipophilic com-
pounds with organic solvents (Tchorbanov and Bozhkova,
1988).
Dierent proteolytic enzymes (from animal, plant and
microbial origin) can be used for production of protein
hydrolysates from algae extracted with ethanol (Fig. 1).
Stoilov et al. (1995) reported the formation of nely sus-
pended particles consisting of lipids and nucleic acids from
the hydrolysed algae (Spirulina pacica with the alkaline
protease subtilisin DY), stabilized by surfactants (i.e. gly-
colipids). The results in our study indicate that cell proteins
are more readily hydrolysed after extraction with ethanol.
One possibility is that the removal of the lipophilic sub-
stances improves the enzyme-substrate contact. The best
results in terms of the degree of hydrolysis were reached
with pancreatin and papain, followed by trypsin, brome-
lain and the Bacillus preparation. Hydrolysates with a
degree of hydrolysis higher than 10% can be used in nutri-
tional supplements and medical diets (Vioque et al., 2004).
Pepsin hydrolyses only a small portion of Chlorella protein,
presumably due to the adoption by the algal protein of an
ionization state, which could be critical for solubilization
20
a
Degree of hydrolysis (%)
15
b
10
5 c
0
sin ai
n
ps
in
p p
pe pa try
br
o B.
Fig. 1. Degree of hydrolysis of cell proteins in ethanol extracted Chlorella
biomass with dierent proteases. A 10% suspension in water of extracted
biomass was hydrolysed (20 U/g) at 37 C for 4 h, accompanied by
continuos stirring. The pH was adjusted to the optimum for each
enzymatic preparation. Each value is the mean of three experiments.
Means without the same letter above bars are signicantly dierent at the
5% level according to the StudentNewmanKeuls test.
H.J. Morris et al. / Bioresource Technology 99 (2008) 77237729
and/or for the interaction with the protease at its optimum
pH.
In view of the high degree of hydrolysis, we chose pancre-
atin for further experiences. In accordance with the stated
above, the degree of hydrolysis of cell proteins was 2-fold
in ethanol extracted biomass (Fig. 2). The utilisation of pan-
creatic enzymes, alone or combined with other proteases,
has been widely reported in protocols for food protein
hydrolysis, such as: casein (Boza et al., 1995), sunower iso-
lates (Megias et al., 2004), whey proteins (Lara et al., 2005),
soy (Lo and Li-Chan, 2005) and wheat gluten (Kong et al.,
2007).
Enzymatic hydrolysis of algae cell proteins depends on
the initial enzyme concentration as well. Independently of
[E0] the enzymatic hydrolysis presented two major stages
(Fig. 3). During the rst two hours the hydrolysis could
account for the soluble algae proteins. Afterwards, the
insoluble proteins inside the cells presumably come into
contact with the enzyme molecules. The globular structure
of the major proteins of Chlorella biomass could be per se
an important limitation on the action of proteolytic
enzymes. This fact has been reported by Clemente et al.
(1999) in the production of chickpea protein hydrolysates.
Moreover, the enzyme inhibition and/or inactivation is an
aspect that has limited the advance to reach higher ecien-
cies in traditional technologies (Guadix et al., 2000; Morris,
2000). An enzyme concentration of 3.04.5 U/mL (30
45 U/g of algae) appeared to be optimal as the degree of
hydrolysis went up to 2022%. The further increasing of
25
a
20 a
Degree of hydrolysis (%)
a
15
a
a
10 b
b b
b
b
a
5
b
s 0
ai
n
tili at
in
el b e 0 1 2 3 4
m su cr
pa
n
Time of hydrolysis (h)
Fig. 2. Changes of the degree of hydrolysis during the enzymatic
hydrolysis of ethanol extracted (s) and non-extracted algae () by
pancreatin (30 U/g). Suspensions in water (10%) of extracted and non-
extracted algae were hydrolysed at pH 7.5 and 50 C for 4 h, accompanied
by continuos stirring. Each value is the mean of three experiments. For
each time of evaluation, means without the same letter are signicantly
dierent at the 5% level according to the MannWhitney test.
 H.J. Morris et al. / Bioresource Technology 99 (2008) 77237729 7727

30 30 200
a a

b
Degree of hydrolysis (%)

Amino nitrogen (mg/ 100 mL)

b

Soluble hydrolysed protein

a
a
a
25 180
a b
b aa
a
ab ab
20 a ab
20 b b 160
ab b b
ab b b

(mg/mL)
a b
ab
b
b
c c c
a ab bc c
15 140
c
bc c
ab
10 a bc c d
c c d
bc d 10 120
d
d
ab c d
c
c
c
b 5 100
0
0 60 120 180 240 e
0 80
t (min) 0 2 4 6 8 10
E0 (U/mL) pH
0.55 1 2 3 4.5
Soluble hydrolysed protein Amino nitrogen
Fig. 3. Hydrolysis curves of Chlorella cell proteins in ethanol extracted
biomass at dierent initial enzyme concentrations. Suspensions in water Fig. 4. Inuence of pH on the concentration of amino nitrogen and
(10%) of extracted algae were hydrolysed with pancreatin at pH 7.5 and soluble hydrolysed protein during enzymatic hydrolysis of ethanol
50 C for 4 h, accompanied by continuos stirring. Each value is the mean extracted algae by pancreatin (30 U/g). Suspensions in water (10%) of
of three experiments. For each time of evaluation, means without the same extracted algae were hydrolysed with pancreatin at 50 C for 4 h,
letter are signicantly dierent at the 5% level according to the Student accompanied by continuous stirring. Each value is the mean of three
NewmanKeuls test. experiments. Means without the same letter are signicantly dierent at
the 5% level according to the StudentNewmanKeuls test.

the amount of the enzyme used did not lead to signicant

higher levels of amino nitrogen and soluble hydrolysed The results of the inuence of pH on pancreatin action
protein (data not shown). are presented in Fig. 4. The highest concentration of
The concentration of the algae suspension is another amino nitrogen and soluble hydrolysed protein were
factor for the optimization of Cv-PH yield. The increase reached at pH values of 7.58.0, which correspond to
of algae concentration was associated with higher values the optimum pH of proteolytic enzymes contained in pan-
of amino nitrogen and soluble hydrolysed protein (Table creatin. The pH of the reaction medium inuences Cv-PH
3). A concentration ranging from 10% to 15% appeared yields. Some non-hydrolysed protein fragments with iso-
to be optimal, since the yield of Cv-PH was much lower electric point within the pH range of 45 would precipitate
with the algae concentration of 20%, even at an eective at this interval. Moreover, undesired changes in the amino
enzyme-substrate ratio, due to complications at the separa- acids can occur under extreme alkaline conditions. Thus,
tion stage. The suspension formed, fails to clarify upon the pH dened in our study was suitable to avoid these
centrifugation, and clear solutions can be obtained only disadvantages.
after ultraltration. The chemical composition of the microalgal products is
the most important factor to evaluate their potential for
utilisation as food supplements. The proximate composi-
tion of Cv-PH is given in Table 2. The in vitro protein
Table 3
digestibility (higher than 95%) could be considered suitable
Inuence of the algae slurry concentration on the amino nitrogen, soluble
hydrolysed protein and yield of protein hydrolysate* for the purpose of the present paper. The bulk of the nitro-
gen fraction consists of soluble hydrolysed protein (42.0%)
Algal slurry Amino Soluble hydrolysed Yield of protein
concentration nitrogen protein (mg/mL) hydrolysate (%)** and free amino acids (5.7%). The amino nitrogen content
(%, w/v) (mg/100 mL) was to be 2.10%, whereas the total nitrogen reached
5 120 8c 13.4 1.5c 32 3.6b 7.95% for the hydrolysates prepared. This ensured an
10 185 5b 24.7 2.0b 52 2.6a amino nitrogen/total nitrogen rate of 26.4%, appropriate
15 190 10ab 31.4 2.5b 52 2.9a for protein solubilization and assimilation. The high solu-
20 205 8a 39.1 1.8a 39 3.2b ble sugars and ash content came from Chlorella biomass.
Each value is the mean of three experiments. Means without the same Enzymatic hydrolysis was strongly preferred over chem-
letter are signicantly dierent at the 5% level according to the Student ical methods for producing microalgae protein hydroly-
NewmanKeuls test.
* sates. It was carried out under mild biological conditions
Suspensions in water of extracted algae were hydrolysed with pan-
creatin (30 U/g) at pH 7.5 and 50 C for 4 h, accompanied by continuos so that the overall amino acid composition of Cv-PH was
stirring. similar to that of the starting material, maintaining its
**
In relation to the extracted algae. nutritional value. The mean amino acid composition of
7728 H.J. Morris et al. / Bioresource Technology 99 (2008) 77237729

Table 4 Cv-PH did not show signicantly dierent values with

Amino acid composition and chemical score of biomass and protein respect to the starting materials.
hydrolysate (Cv-PH) of Chlorella vulgaris 87/1*
Molecular weight patterns of an aqueous hot-extract
Amino acid Non- Ethanol Cv-PH FAO from ethanol extracted Chlorella biomass and Cv-PH were
extracted extracted patterna
biomass biomass
determined by gel ltration chromatography in a Sephadex
G-100 column (Fig. 5). The proles of extracts from etha-
Histidine 2.18 0.25 2.14 0.12 2.10 0.09 1.9
Isoleucine 4.49 0.60 4.52 0.70 3.80 0.10 2.8
nol treated algae were characterized by the presence of six
Leucine 9.80 0.31 10.08 0.25 9.20 0.38 6.6 major protein fractions, with molecular masses ranging
Lysine 7.10 0.24 7.00 0.33 6.90 0.35 5.8 from 120 to 12 kDa. As a result of the hydrolysis, Cv-PH
Methionine + 1.92 0.09 1.80 0.16 1.80 0.12 2.5b showed a major chromatographic peak of molecular mass
Cysteine lower than 10 kDa (Cv-PHB). Cv-PH showed total disap-
Phenylalanine + 7.84 0.24 7.62 0.11 7.50 0.16 6.3c
Tyrosine
pearance of the protein fraction with molecular mass about
Threonine 4.56 0.38 4.45 0.17 4.30 0.32 3.4 28 kDa. The eectiveness of hydrolysis was minor with
Valine 7.86 0.26 7.41 0.33 8.0 0.49 3.5 fractions of 19 and 12 kDa. The ltration of Cv-PHB
Tryptophan 1.15 0.05 1.10 0.02 1.10 0.03 1.1 through Sephadex G-25 gel allowed the identication of
Alanine 11.47 0.15 11.43 0.09 11.20 0.24 three main peptides with molecular masses between 2 and
Arginine 6.0 0.28 6.0 0.19 5.70 0.25
Aspartic acid 10.14 0.18 10.40 0.22 10.60 0.33
Glutamic acid 14.35 0.06 14.30 0.05 14.30 0.02
Cv-PHB
Glycine 5.26 0.36 5.05 0.28 5.20 0.24 0.8 28 kDa
Proline 5.16 0.24 4.88 0.22 5.10 0.18 19 kDa
Serine 3.30 0.17 3.50 0.32 3.20 0.15 69 kDa
0.6 120 kDa 12 kDa
Essentials/total 46.9 45.3 44.7
A 214 nm

(%) 15 kD
Chemical score 0.77 0.72 0.72 0.4
*
Each value is the mean SE of three determinations (g of amino acid/
100 g protein). 0.2
a
FAO/WHO/UNU (1985).
b
Met + Cys. 0
c
Phe + Tyr. 0 10 20 30 40 50 60 70
Volume eluted (mL)

Cv-PH is presented in Table 4, in comparison to the FAO/ Cv-EA Cv-PH

WHO amino acid requirement pattern for the 25 year old
child to be used as the reference protein (FAO/WHO/
UNU, 1985). Amino acid data indicate that C. vulgaris
87/1, both ethanol extracted and non-extracted, contains
adequate amounts of essential amino acids in relation to
the reference pattern and constitutes a suitable protein
source for the elaboration of protein hydrolysates. The
exception is the low content of S-containing amino acids
(Met and Cys), which is also very common in other micro-
organisms (Becker, 1994). The chemical score used to esti-
mate the quality of Cv-PH was 0.72 for sulfur amino acids
(limiting amino acids), that makes the availability of Met
Fig. 5. Gel ltration chromatography in Sephadex G-100 of Chlorella
vulgaris extract (Cv-EA) and pancreatic protein hydrolysate (Cv-PH). The
column (1  60 cm) was eluted with phosphate buer 0.1 mol/L pH 6.8 at
a ow rate of 12 mL/h. Molecular weights of major protein fractions,
determined from the calibration of the column with molecular weight
standards are showed. The peak Cv-PHB was collected for further
fractionation in Sephadex G-25 gel.
0.6
3500 Da
0.4
A 214 nm

and Cys in the protein hydrolysate from the algae strain 2000 Da
C. vulgaris 87/1, used in this investigation, very important.
Methionine is somewhat labile to acid hydrolysis (Bucci 4600 Da

and Unlu, 2000). The values reported in Table 4 for methi- 0.2
onine thus underestimate the actual methionine concentra-
tion in the protein of this microalgae. In contrast, lysine,
the most limiting essential amino acid in cereals, is present 0
in the hydrolysate in concentrations that surpassed the 0 5 10 15 20
FAO reference protein. This fact suggests the possibility Volume eluted (mL)
of supplementation of other protein sources with Cv-PH.
With respect to the essential amino acid to total amino Fig. 6. Gel ltration chromatography in Sephadex G-25 of the low
molecular peak of Chlorella vulgaris protein hydrolysate (Cv-PHB)
acid ratio (E/T%), the value of 44.7% in Cv-PH is clearly
obtained in Sephadex G-100 chromatography. The column (1  60 cm)
above the third of the total amino acid content, which is was eluted with phosphate buer 0.1 mol/L pH 6.8 at a ow rate of
an index of the nitrogenous equilibrium according to the 12 mL/h. Molecular weights of major peptides, determined from the
nutritional recommendations (FAO/WHO/UNU, 1985). calibration of the column with molecular weight standards are showed.
 H.J. Morris et al. / Bioresource Technology 99 (2008) 77237729
5 kDa (Fig. 6). This proteolytic modication could have
special importance for the improvement of solubility of
algal protein and for decreasing its residual antigenicity.
In conclusion, the utilisation of C. vulgaris 87/1 cell bio-
mass derived from outdoor cultivation in tropical condi-
tions for the production of enzymatic protein hydrolysates
has been reported. Cv-PH was characterized by its high pro-
tein quality, mainly balanced amino acid content and a high
in vitro protein digestibility, which accounted for its possible
use as an additive for special foods. In this sense, Morris
et al. (2007) have recently reported the immunostimulant
activity of Cv-PH on undernourished mice.
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