World Journal of Microbiology & Biotechnology 16: 563565, 2000.

563
2000 Kluwer Academic Publishers. Printed in the Netherlands.

Eect of Spirulina platensis biomass on the growth

of lactic acid bacteria in milk

Gloria Zulpa de Caire1,*, Jose Luis Parada2, Mar a Cristina Zaccaro1 and Monica M. Storni de Cano1
1
Biologa de Cyanobacterias, Depart. Ciencias Biologicas, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria, Pab. II, (1428), Buenos Aires, Argentina
2
Laboratorio de Microbiologa de Alimentos, Depart. Qumica Organica, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria, Pab.II, (1428), Buenos Aires, Argentina
*Author for correspondence: Tel.: 54 11 4576 3349, Fax: 54 11 4576 3384, E-mail: cyanob@bg.fcen.uba.ar

Received 3 March 2000; accepted 25 August 2000

Keywords: Bioactive compounds, cyanobacteria, lactic acid bacteria, Spirulina platensis

Summary

The stimulatory eect of aqueous suspensions of Spirulina platensis dry biomass extracted at pH 6.8 and 5.5 was
studied on four lactic acid bacteria (LAB) grown in milk. The addition of dry S. platensis to milk (6 mg/ml)
stimulated growth of Lactococcus lactis by 27%. The growth of other strains was also promoted.

Introduction Materials and Methods

Yoghurt and other fermented milks contribute to health Strains

with natural nutrients and enrich the intestinal ora
with lactic acid bacteria (LAB). They provide humans
with major benets: protection from infection (Nader de
Mac as et al. 1993), stimulation of the immune system
(Perdigon et al. 1992), as well as better digestion and
absorption of lactose and minerals (Hove et al. 1999).
Cyanobacteria or bluegreen algae are photoauto-
trophic micro-organisms widely distributed in nature.
They have been used as human food for centuries.
Spirulina sp. is the best known genus because of its
nutritional value. It contains 18 of the 20 known
aminoacids; high quality proteins; more calcium than
milk; more vitamin B12 than cow liver; vitamins A, B2,
B6, E, H and K, and all essential minerals, trace
elements and enzymes (Fox 1986). Spirulina is one of the
richest sources of iron among various organic health
supplements. The iron over twice as absorbable as the
form found in vegetables and most meats. The fatty acid
composition of Spirulina is characterized by high levels
of the x-6 series. Deciency in linolenic acid is also
associated with vision (Neuringer & Connor 1986) and
nervous system defects, regulation of blood pressure,
cholesterol synthesis, inammation and cell prolifera-
tion. Recently, we observed that growth of LAB in
synthetic media was promoted by Spirulina platensis
extracellular products (Parada et al. 1998).
The objective of this work was to study the eect of a
natural additive, dry biomass from S. platensis, on the
growth of LAB in milk.
Cyanobacteria: S. platensis 945. LAB: Streptococcus
thermophilus TH4, Lactobacillus delbruekii subsp. bul-
garicus YL1, L. lactis subsp. lactis C2, and Lactobacillus
acidophilus LO1.
Culture media
Zarrouk culture medium (Parada et al. 1998) was
prepared for S. platensis using proanalysis reagents
Merck or equivalent (pH 11). For LAB, MRS broth
and agar media (Merck) and Nestle dried skimmed
reconstituted milk were used.
Experimental design
Spirulina platensis was cultured in Zarrouk medium at
28 2 and 45 lEinstein/m2/s light intensity. The
biomass was separated by ltration when the culture
reached the stationary phase and was washed with acid
water until pH 7 was obtained. The biomass was dried at
30 C and made into powder. The LAB inocula were
grown in MRS broth.
Two suspensions of S. platensis were prepared, one in
distilled water at pH 6.8 and the other in buer
phosphate 0.1 M pH 5.5, and kept in refrigeration for
24 h. They were then added to reconstituted dried
skimmed milk 10% (w/v) and treated at 100 C for
20 min.
564 G.Z. de Caire et al.
LAB TH4, YL1 and C2 were cultured at 37 C in milk
plus a suspension of S. platensis to a nal concentration
of 3 mg/ml (pH 6.8) and strains C2 and LO1 were
grown in milk at a nal concentration of 6 mg/ml
(pH 5.5). In both cases controls were prepared without
the addition of S. platensis.
Samples were taken at dierent periods of time to
establish the growth kinetics by counting colonies on
MRS agar and the results were expressed as c.f.u./ml.
For each experiment six replicates were prepared.

Statistical methodology

Analysis of variance was performed for all data, using a

complete randomized experimental design. The homo-
geneity of the mean squares of the experimental error
was assessed by the Bartlet Test. Duncan Test was used
for the comparison of means at a level of signicance of
P < 0.05, ``Statistics'' PC program (Steel & Torrie
1980).

Results and Discussion

To evaluate the eect of S. platensis dry mass on LAB

growth in milk S. thermophilus TH4, L. lactis C2 and
L. delbruekii YL1 were grown with and without the
addition of 3 mg of S. platensis/ml. After 4 h the LAB
growth promotion by S. platensis, at pH 6.8, was
13.42% for C2; 9.29% for YL1 and 8.22% for TH4,
compared with the controls. After 8 h the increase was
3.46, 9.73, 7.76% and for C2, YL1 and TH4 respective-
ly, probably due to a decrease in the amount of the
stimulatory factors. The three strains treated with
Spirulina reached the stationary phase after 10 h and
the counts remained the same up to 20 h, while the same
strains without Spirulina addition grew more slowly and
they continued up to 20 h, reaching the same value as
the supplemented ones (Table 1).
The eect on C2 and LO1 added with twice the
amount of S. platensis, pH 5.5, is shown in Figure 1.
The growth promotion of C2 was 27.3% after 4 h and
for strain LO1 an increment of 22.8% was observed
after 8 h.
The tested LAB showed more growth in milk enriched
with natural nutrients from the Spirulina, and they
clearly responded to dierent extents according to the
strain.
Figure 1. Growth kinetics of LO1 (r,e) and C2 (.,,) without and
with 6 mg/ml Spirulina plantensis (n = 6).
Parada et al. (1998) found that addition of extracellular
products obtained from a late log phase culture of
S. platensis promoted the growth of some LAB. On this
account it was suggested that S. platensis could have a
stimulatory eect on LAB by acting as a prebiotic
substance (Gibson & Roberfroid 1995).
Spirulina platensis dry mass (6 mg/ml) would make
an additional contribution to 220 ml of fermented milk
of: proteins, 855 mg; provitamin A, 0.75 mg (RDA
3.05 mg); rivoavin (B2), 0.05 mg (RDA, 1.70 mg);
pyridoxine (B6), 0.002 mg (RDA, 2 mg); cyanocobal-
amin (B12), 3.6 lg (RDA, 6 lg); biotin (H), 0.01 lg
(RDA, ND); phosphorus, 20 mg (RDA, 1000 mg); iron,
2 mg (RDA, 18 mg); linolenic acid, 15.55 mg (RDA,
ND); c-linolenic acid, 20.25 mg (RDA, ND) and mu-
copolysaccharide, according to the chemical composi-
tion tables given by Richmond (1988) and Henrikson
(1994).

Table 1. Growth kinetics of lactic acid bacteria (c.f.u./ml) added with 3 mg/ml of S. platensis, and corresponding controls (n = 6).

0h 2h 4h 8h 10 h 20 h

TH4 4.602 0.115 4.670 0.212 5.544 0.221 6.146 0.124 7.322 0.651 7.851 0.611
TH4 + S. platensis 4.602 0.212 4.819 0.212 6.000 0.110 6.623 0.099 7.898 0.711 7.897 0.655
C2 4.892 0.151 4.892 0.203 5.857 0.154 6.633 0.115 6.756 0.455 7.531 0.545
C2 + S. platensis 4.892 0.302 5.146 0.100 6.643 0.151 6.860 0.115 7.398 0.600 7.491 0.400
YL1 4.785 0.204 4.819 0.201 5.623 0.201 6.505 0.209 7.462 0.354 7.643 0.668
YL1 + S. platensis 4.785 0.145 4.965 0.301 6.146 0.101 7.138 0.203 7.724 0.200 7.755 0.740
Spirulina platensis and lactic acid bacteria
Nowadays, when the dairy industry is supplementing
milk with minerals, vitamins and antioxidants, it would
be of interest to consider the possibility of adding
Spirulina biomass, as a natural product, to fermented
milk to induce a faster production of LAB and
increase the number of viable cells in the product
and in the gut.
Acknowledgements
The authors thank to Universidad de Buenos Aires-
Ciencia y Tecnica for support.
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