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CHAPTER - V
RESULTS AND DISCUSSIONS
5.1 INTRODUCTION
The main aim of this thesis is to produce high quality Spirulina with affordable
price for common peoples. In Chapter 4 all the results are tabulated for producing
Spirulina at mass production level. In this chapter we discuss the results by plotting
graph by using various parameters.
5.2 EVALUATION AND ENHANCEMENT OF ANTIOXIDANT IN SPIRULINA
Evaluation and enhancement of antioxidant properties of Spirulina are done in all
studies.
5.2.1 Study 1
Figures 5.1 to 5.5 show the growth curves for the four different conditions of
growth using identical nutrients. The highest growth was achieved with those operations
without any artificial light treatment. During summer at peak time, partial shading may
engender better growth and normal sunny days it does not required. Here control gives a
favorable response in terms of production, quality and yield. Full time operation of
agitation and light source has not produced any significant result. So, it is concluded that
the best operation time for agitation is between 7.00 Am and to PM. Providing shadow
(shadenet) also has not given any satisfactory results during normal time and it given
favorable response when atmospheric temperature is above 35oC during hot summer
days.
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Fig 5.1 Optical Density vs Days Jan 14
Fig 5.2 Optical Denisity vs Days April 14
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Fig 5.3 Total Carotenoids vs week Jan 1
Fig 5.4 Total Carotenoids vs week April 14
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Fig 5.5 Chlorophyll vs week Jan 14 Fig 5.6 Chlorophyll Vs week Apr 14
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Fig 5.7 phycocyanin Vs week Jan 14
Fig 5.8 Phycocyanin Vs week Apr 14
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Fig 5.9 Pigments Vs week Jan & Apr 14
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5.2.2 Study II
Results based on the trials indicate that any one system is required when control
was shown higher bacterial count. Adoption of good practices can help a maximum
reduction of 10 to 15% and with some technique for controlling bacterial growth. During
this study we observed that some treatment helped eradication of bacterial count and
some techniques can make microbes inactive (dormat stage) and with a suitable
environment bacteria can see vigorous multiplication spoiling the product in the process.
UV radiation, Ozonization gave good performance during experiments and storage time
it was less.
Sterlization: It is a good system for controlling microbes and does eradication
from products also under high pressure and temperature. No increase doing storage.
Sanitation: This technique too gives good response but has to be taken up only
during the intermediate stage (wetmass).
We suggest control of bacterial count through use of both sanitation and
sterilization processes in Spirulina production. Use of Hypo chlorite solution at 20PPM
for biomass washing is recommended as also sterilization during the power stage.
Graph 5.10 to 5.14 describes the control of bacterial count on as follows.
UV radiation: We have tried with different time intervals from 10 minutes to 60
minutes, resulted lower bacterial count at 50 minutes and 60 minutes radiation.
Ozonization: we have made trail with two time interval with three different
concentrations. Here, controlled microbed at 4ppm and 6 ppm of 30 miuntes.
Sterlization: This system developed specifically for Spirulina, it gave good
response. This system also gives control in storage. Trail was taken in 121 & 134oC at
different time intervals. We got a lower count at 134oC.
Chlorination:
Sodium hypo chlorite for chlorination was used and it was favoured at higher 20 PPM.
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Fig 5.10 UV Radiation - control Vs treated Fig 5.11 Ozonization – control Vs Treated
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Fig 5.12 Heat sterilization - Control Vs Treated
Fig 5.13 Chlorination – control Vs Treated
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5.2.3 Study III
Based on this study commercial medium and modified inorganic medium gave
better results than the organic medium. Slow growth and lower yield are seen with use of
organic medium due to inadequate nutrients source (NPK). Commercially available
organic source is not helpful when compare to a commercial non organic medium.
Further study is required for use of different organic inputs with different ratios for
improving the growth of Spirulina. About 70 % of production was seen when compared
to a non organic medium but the premium value of Rs.1300/kg and Non organic at
Rs.800/kg. Nonorganic input is better than organic one with reference to the value of
product.
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Fig 5.14 Optical Density Vs Days
Fig 5.15 pH Vs Days
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Fig 5.16 Dry weight l Density Vs Days
Fig 5.17 Protein Vs week
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5.2.4 STUDY IV
Raw herbs (Indian gooseberry) and Spirulina were estimated separately. Both
were studied after experiments and study showed that vitamin C level increased in
Spirulina. Antioxidant activity also favourably increased compared to non treated
Spirulina. Normally, Spirulina does not contain vit C in its profile.
Finally, this thesis was reports optimum temperature, agitation time, system for controlling microbial count, low cost medium and vit C/selenium enriched Spirulina
towards increasing antioxidant activity. It is understood that Pigments and vitamins control the anti oxidant activity of Spirulina and mass production depends on
temperature, aeration, inputs. Providing uniform temperature and proper agitation,
sterilization and enriching vitamin C to the products can achieve high anti oxidant
activity Spirulina when compared to normal Spirulina. This has been proved by
analyzing the pigments and vitamin C level in Spirulina.
Test was carried out with commercial production of Spirulina. Two percentage of
ascarboic acid is added during the process. This is extracted from Indian gooseberry by
aqueous technique and the same was mixed in proportionate ratio before spray drying.
The spray dried Spirulina powder was collected thro drier rotary valve and the same was
examined. Availability of vitamin C and antioxidant property was measured by using
DPPH method. It was tabulated and made graph between antioxidnat property and amla
juice ration. We found that the satisfied quantity of vitamin C as per human consumption
was arrived at nearly 20 mg/gm of Spirulina powder.
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Fig 5.18 Antioxidant activity Vs week Apr 14
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5.3 NUTRITIONAL VALUE OF SPIRUINA Vitamins & Enzymes
Vitamin A(Beta-Carotene) 15,030 iu
Vitamin B1(Thiamin) 76 mcg
Vitamin B2(Riboflavin) 104 mcg
Vitamin B3(Niacin) 466 mcg
Vitamin B6 14 mcg
Vitamin B12 7.4 mcg
Vitamin E(d-a tocopherol) 106 mcg
Inositol 2.0 mcg
Biotin 0.969 mcg
Folic Acid 0.9 mcg
Pantothenic Acid 12 mcg
Superoxide Dismutase (S.O.D) 2600 units Carotenoids/Phytonutrients
Beta-carotene 9-cis 1.6 mg
Beta-carotene 13-cis .51 mg
Beta-carotene 15-cis .12 mg
Beta-carotene all-trans 7.8 mg
Zeaxanthin .95 mg
Total carotenoids 13 mg
Chlorophyll 21 mg
Phycocyanin-crude 200-350 mg
C-Phycocyanin 90-160 mg
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Minerals
Calcium 8.4 mg
Magnesium 11 mg
Iron 1.7 mg
Phosphorus 30 mg
Potassium 59 mg
Sodium 60 mg
Manganese 115 mcg
Zinc 70 mcg
Boron 30 mcg
Copper 15 mcg
Molybdenum 7.5 mcg
Selenium 7.8 mcg Fatty Acids
Gamma Linolenic(GLA) 20-30 mg
Essential Linolenic 33 mg
Dihomogamma Linolenic 1.59 mg
Alpha Linolenic 0.0435 mg
Palmitoleic 5.94 mg
Oleic 0.51 mg
Erucic 0.072 mg
Palmitic Acid 60 mg
Myristic Acid 1.24 mg
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Stearic Acid 0.204 mg
Arachidic Acid 0.144 mg
Behenic Acid 0.144 mg
Lignoceric Acid 0.072 mg
The following eight essential amino acids are found in Spirulina:
ISOLEUCINE: It is required for optimal growth, intelligence development and
nitrogen equilibrium in the body . It is also used to synthesize other non-essential
amino acids.
LEUCINE: It is a Stimulator of brain function which increases muscular energy
levels.
LYSINE: It is used to build block of blood anti bodies, strengthens circulatory
system and maintains normal growth of cells.
METHIONINE: Vital lipotropic (fat and lipid metabolizing) amino acid that
maintains liver health. It also acts as an anti-stress factor, it calms the nerves.
PHENYLALANINE: It is required by the thyroid gland for the production of
thyroxine which stimulates metabolic rate.
THREONINE: It improves intestinal competence and digestive assimilation.
TRYPTOPHANE: It increases the utilization of B vitamins, improves nerve
health and stability of the emotions. It promotes sense of calm.
VALINE: It stimulates mental capacity and muscle
coordination. Non essential amino acids:
ALANINE: Strengthens cellular walls,
ARGININE: Important to male sexual health as seminal fluid is 80% arginine.
Also helps to detoxify the blood.
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ASPARTIC ACID: Aids transformation of carbohydrates into cellular energy.
CYSTINE: Aids pancreatic health, which stabilizes blood sugar and
carbohydrate.
GLUTAMIC ACID: With glucose, one of the principal fuels for the brain cells.
Has been used to reduce the craving for alcohol and stabilize mental health.
GLYCINE: Promotes energy and oxygen use in the cells.
HISTIDINE: Strengthens nerve relays, especially in the auditory organs. Has
been used to reverse some cases of deafness.
PROLINE: A Precursor of glutamic acid
SERINE: Helps from the protective fatty sheaths surrounding nerve fibers.
TYROSINE: Slows aging of cells and suppresses hunger centers in the
hypothalamus.
Chemistry
Spirulina is composed of approximately
Crude protein 61 – 65 % &
Carbohydrates 18 – 22 %
The protein content includes 22 essential amino acids and the total protein is
nutritionally superior to legume protein, but inferior to meat protein. The proteins C-
phycocyanin and allophycocyanin in Spirulina have been the focus of much research.
It contains the high levels of B-complex vitamins, vitamin E, beta-carotene and
zeaxanthin.
High levels of gamma linolenic acid and a polyunsaturated fatty acid contain 300
to 400 ppm iron (dry weight) and unlike many forms of plant iron, has high
bioavailability when ingested by humans.
Trace elements present at high levels include manganese, selenium and zinc.
Calcium, potassium and magnesium are also concentrated in the organism.
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Uses and Pharmacology
Clinical trials are investigated Spirulina's potential but it has been too small to
support its purported effects.
Allergic and asthma
Experimental data have suggested that C-phycocyanin can selectively inhibit
release of histamine from mast cells and prevent increases in immunoglobulin E. A small
study in patients with mild to moderate asthma suggested that Spirulina supplementation
(1g/day) produced improvement in lung function parameters while a study evaluating
Spirulina in allergic rhinitis suggested a positive effect on laboratory values, but no
clinical outcomes were reported. A 6-month, double-blind, placebo-controlled study
enrolling 150 patients with allergic rhinitis found efficacy for 2 g/day of Spirulina over
placebo using diary-based symptom scores of nasal discharge.
Antimicrobialactivity
A provocative suggestion has been made that human cultures in which large
amounts of algae are consumed have lower levels of HIV infection. Spirulina and its
extracts have been evaluated for antiviral activity. One in vitro study found that the
sulfated polysaccharide calcium spirulan interfered with the replication of several
enveloped viruses, including herpes simplex, cytomegalovirus, mumps, measles,
influenza A and HIV-1, while another study described a slightly different range of
viruses susceptible to the extract. HIV-1 adsorption and penetration were inhibited by an
aqueous extract of Spirulina, while a crude hot water extract reduced HIV-1 replication.
This type of in vitro activity is common to acidic polysaccharides from a variety of
sources. Enterovirus is also susceptible to Spirulina and allophycocyanin was the active
constituent. Spirulina demonstrated some in vitro activity against common human
bacterial pathogens but less than that of the standard comparator. Immune stimulation by
phycocyanin and polysaccharides of Spirulina led to an antifungal and antibacterial
effect in mice.
Cancer
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C-phycocyanin showed a dose-dependent inhibition of HeLa and human chronic
myeloid leukemia cell growth and proliferation in vitro. Induction of apoptosis was
considered to be one of the mechanisms involved. Doxorubicin-resistant HepG2 liver
cancer cells were inhibited by Spirulina C-phycocyanin through an apoptotic
mechanism, while water-soluble polysaccharides were implicated as the active agent
against stomach cancer cells. A combination of selenium and Spirulina inhibited MCF-7
breast cancer cells via growth arrest and apoptosis. Survival rates increased in mice with
liver cancer treated with C-phycocyanin, and tumor regression has been reported in
animals with oral cancer. Activation of antitumor natural killer cells by Spirulina
enhanced antitumor efficacy in a B16 mouse melanoma model, and the effect was
abolished in MyD88 null/null mice, indicating that NK cell activation was a key
pathway. In a hamster cheek pouch model of carcinogenesis, 10 mg/day of Spirulina
extract reduced dysplastic changes, which were further confirmed by an immune
histochemical study. Spirulina was chemopreventative in a dibutyl nitrosamine
carcinogenesis model. It also induced lesion regression in tobacco chewers with oral
leukoplakia in a study conducted in India.
Diabetes
A study in alloxan-induced diabetic rats revealed that Spirulina at 10 mg/kg
orally for 30 days lowered glucose levels, while slightly elevating insulin. Two small
clinical studies investigated the effects of Spirulina supplementation in type 2 diabetes,
with improvement noted in fasting blood sugar and lipid profiles. Suggested mechanisms
of action include hypoglycemia caused by fiber content or possible insulin-stimulating
action of peptides and polypeptides of Spirulina proteins. The actions on lipids have
been attributed to gamma linolenic acid content.
Dietarysupplement
Spirulina, considered a food item for centuries in many countries, is now
popularly thought of as a dietary supplement. Spirulina consumption was purported to
aid in weight loss because of its high phenylalanine content, but a US Food and Drug
Administration review found no evidence to support this claim. Suggestions that
Spirulina is a valuable source of vitamin B 12 have been similarly disputed. Skeletal
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muscle protein (myosin) was increased in young rats fed Spirulina as the sole dietary
protein source compared with casein. A study of Spirulina supplementation for 8 weeks
demonstrated clinical improvement in weight gain and increased hemoglobin levels in
malnourished children in the West African nation of Burkina Faso. Similar results have
been demonstrated among children who are HIV-positive. In elderly Koreans, Spirulina
8 g/day for 16 weeks had a variety of positive effects (cholesterol, antioxidant status,
interleukin [IL]-2 and IL-6 levels) observed in a randomized, double-blind, placebo-
controlled study. In a study of athletic training, Spirulina increased time to fatigue,
decreased carbohydrate oxidation rate, and increased fat oxidation rate, leading to an
increase in exercise performance.
Hyperlipidemia
Experiments in rats suggest that C-phycocyanin exhibits hypercholesterolemic
action. In rabbits fed a high-cholesterol diet, Spirulina (1% or 5% in diet) lowered serum
triglycerides, total cholesterol, and low-density lipoprotein (LDL) at 8 weeks. High-
density lipoprotein (HDL) was markedly increased. Two small clinical studies examined
the role of Spirulina in hyperlipidemia secondary to nephrotic syndrome. Both
populations showed an improved lipid profile with Spirulina supplementation; however,
the control group in 1 experiment also showed improvement. The gamma linolenic acid
content of Spirulina may have played a role in the mechanism of action. A study in type
2 diabetes patients reported a reduction in triglycerides with 8 g/day of Spirulina. In
normal volunteers, 4.5 g/day of Spirulina for 6 weeks lowered blood pressure, total
cholesterol, LDL and increased HDL.
Immune system effects
Most in vitro and animal experiments have suggested immunostimulatory effects;
however, 1 study found a Spirulina extracts to be immunosuppressive. Activation of
monocytes and macrophages as well as augmentation of interleukin and interferon
production, have been demonstrated. Intestinal epithelial lymphocytes of aged mice
treated with Spirulina were increased compared with the control aged group. A clinical
study in healthy men found that oral administration of Spirulina for 3 months resulted in
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enhanced interferon production and natural killer (NK) cell capacity. An ex vivo study of
NK cells from Spirulina-treated healthy patients showed increased NK activity, which
was confirmed by a second study in which NK cell and T-cell markers were increased by
Spirulina. A clinical trial in elderly patients showed positive effects on anemia and
immunosenescence after 6 and 12 weeks of supplementation. The immunostimulatory
effects appear to be largely mediated by Spirulina polysaccharides.
Prevention of toxicity due to metals or organic compounds
A 5% Spirulina-supplemented diet prevented carbon tetrachloride-induced fatty
liver in rats. In a case series of 3 patients with nonalcoholic fatty liver disease, 4.5 g/day
of Spirulina for 3 months improved ALT values and lipid profiles. Cadmium toxicity in
rats was reduced by Spirulina, as measured by liver histopathology. Mercuric chloride-
induced oxidative stress in mice was blocked by Spirulina at 800 mg/kg orally for 40
days. Lead acetate damage to rats was minimized by Spirulina via normalizing plasma
and liver lipid levels, as well as via its antioxidant effect. A protein extract and purified
phycocyanin protected neuroblastoma cells from iron-induced toxicity. Spirulina
pretreatment protected mice against acetaminophen and galactosamine-induced liver
damage. Liver and kidney enzyme markers of toxicity were reduced by Spirulina
following 4-nitroquinoline 1-oxide insult to rats. Spirulina decreased cisplatin-induced
nephrotoxicity in rats, an effect attributed to an antioxidant action. Gentamicin-induced
kidney damage in rats was reversed by intraperitoneal Spirulina 1 g/kg daily. In pregnant
mice, teratogenicity due to cadmium was reduced by 125 to 500 mg/kg of Spirulina by
intragastric administration for 17 days of gestation. Mutagenicity of cyclophosphamide
in mice pretreated with Spirulina was reduced. In a small, randomized, placebo-
controlled trial, Spirulina plus zinc increased urinary excretion of arsenic and decreased
arsenic hair-content in people with long-term exposure to arsenic.
Antioxidant
The Spirulina protein phycocyanin in pure form was active in 4 different cell-free
radical-scavenging assays; however, phycocyanin-containing selenium was more
effective. In cellular assays of antioxidant activity, 4 commercial Spirulina preparations
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were also active. Spirulina supplementation of rats did not increase plasma or liver alpha-
tocopherol levels; however, another study reported effective antioxidant activity using
combinations of whey protein and Spirulina. C-phycocyanin from Spirulina reduced
oxidative stress in hamsters fed an atherogenic diet. Similarly, rabbits fed a high-
cholesterol diet were protected from oxidative stress by 4 to 8 weeks of Spirulina in feed
at 1% or 5%. Other studies suggest Spirulina as an antioxidant, but clinical importance
has not been demonstrated, and 1 small clinical study showed Spirulina to be without
effect on plasma antioxidant status.
Other uses
C-phycocyanin inhibited platelet aggregation in ex vivo experiments. In mice
with zymosan-induced arthritis, phycocyanin exerted a scavenging action against
reactive oxygen species and anti-inflammatory activity. Similar experiments with
complete Freund's adjuvant-induced arthritis found 800 mg/kg of oral Spirulina effective
in reducing inflammation. In rats, collagen-induced arthritis was inhibited by 400 mg/kg
of Spirulina. Osteoporosis was inhibited in rosiglitazone-treated rats by 500 mg/kg/day
of oral Spirulina. A rat study showed evidence that Spirulina could protect neural stem
cells and promote their growth ; however, an amyotrophic lateral sclerosis (ALS) support
network did not find the evidence compelling for use in ALS. Spirulina supplementation
(3 g/day) was ineffective against idiopathic chronic fatigue in a small study. Pretreatment
with Spirulina 180 mg/kg orally in a rat cerebral ischemia-reperfusion injury model
reduced neurologic deficits and histological changes. A polysaccharide extract of
Spirulina was antiangiogenic in a mouse corneal model. Spirulina has also been reported
to protect mouse and human bone marrow cells against gamma radiation.
Spirulina and Diabetes. Spirulina has been found to have significant positive
effects on people suffering from type 2 diabetes mellitus. It is reported that Spirulina has
the ability to reduce he fasting blood sugar levels in the body after 6 to 8 weeks of intake.
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Spirulina and Weightloss: Spirulina today is most popular as a therapy for
weight loss. It is mostly marketed now as the most vitamin rich appetite suppressant.
Spirulina is actually one of the non-meat products which contain the highest protein
content, along with all the essential nutrients and amino acids, without the unnecessary
carbohydrates or fats. Your waistline will surely be kept where you want it.
Spirulina and Cholesterol. Based on the same concept as above, Spirulina is
also promising in terms of lowering blood cholesterol and triglyceride levels. Also,
Spirulina is said to contain gamma-linoleic acid (GLA), which is found uniquely on
mother's milk. This essential fatty acid is vital to promoting heart health in general
Comparision between
Commodity Protein Carbohydrate Lipid
Meat 43 1 34
Egg 47 4 41
Milk 26 38 28
Rice 8 77 2
Soya 37 30 20
Chlorella vulgaris 51 17 22
Dunaliella salina 39 14 14
Haematococcus pluvialis 10 40 41
Spirulina platensis 60 18 5 Usable Protein of Common Protein Foods
Food Protein (%) NPU(%) ** Usable Protein
(%)
Dried eggs 47 94 44
Spirulina 65 57 37
Skimmed Milk 36 82 29
Soy flour 37 61 23
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Fish 22 80 18
Chicken 24 67 16
Beef 22 67 16
Peanuts 26 38 10
Switzer (1982) ** Net Protein Utilization
Food Comparision Chart
Food Beta-Carotene
Spirulina, 3 grams 9.0 mg
Carrot, 1 Medium 4.9 mg
Papaya, 1 Medium 3.7 mg
Chlorella, 3 grams 1.0 mg
Apricot, 1 Medium 0.9 mg
5.4 CONCLUSION
In this study gives appropriate techniques for mass production of Spirulina in
open pond system. Developed new novel ideas for controlling microbial contaminations,
cost reduction, optimization of natural resources and enriching vitamin C level in
spirulina.
CHAPTER – VI
CONCLUSIONS AND SCOPE FOR FUTURE WORK
6.1 INTRODUCTION
In this dissertation, four key issues are addressed in a micro algae production
process system: 1.Identify the impact of natural resources and optimize it 2.Reduce the