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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