Ami Ben-Amotz

The biotechnology of cultivating the halotolerant algaDunaliella

Abstract The unicellular alga Dunaliella thrives in media with very high salt concentrations, accumulating large amounts of commercially important chemicals such as β-carotene and glycerol. Dunaliella is cultivated commercially in large outdoor ponds, and harvested to produce high β-carotene dry algal meal and concentrated algal β-carotene in oil, used by the health-food industry and for food-coloring, respectively. The algal β-carotene differs from the synthetically available β-carotene in its stereoisomeric composition and may be of use as a pharmaceutical product.

Bio-diesel production directly from the microalgae biomass of Nannochloropsis by microwave and ultrasound radiation.

This work offers an optimized method for the direct conversion of harvested Nannochloropsis algae into bio-diesel using two novel techniques. The first is a unique bio-technology-based environmental system utilizing flue gas from coal burning power stations for microalgae cultivation. This method reduces considerably the cost of algae production. The second technique is the direct transesterification (a one-stage method) of the Nannochloropsis biomass to bio-diesel production using microwave and ultrasound radiation with the aid of a SrO catalyst. These two techniques were tested and compared to identify the most effective bio-diesel production method. Based on our results, it is concluded that the microwave oven method appears to be the most simple and efficient method for the one-stage direct transesterification of the as-harvested Nannochloropsis algae.

Analysis of carotenoids with emphasis on 9-cis β-carotene in vegetables and fruits commonly consumed in Israel

Abstract Recent epidemiological studies have directed the attention from the synthetic all- trans β-carotene to natural carotenoids predominant in fruits and vegetables as possible active ingredients for prevention of cancer and cardiovascular diseases. Seventeen fruits and 17 vegetables commonly consumed in Israel and the β-car-otene-rich alga, Dunaliella bardawil , were analysed for their content of carotenoids with emphasis on 9- cis β-carotene by reversed-phase, 3D photodiode array HPLC. Fourteen carotenoids were eluted in order of decreasing polarity, from polar oxycarotenoids to lipophilic hydrocarbons, and quantified in μg carotenoid per gram freeze-dried plant sample. The richest sources of total carotenoids (>100μg/g dry weight) in Israeli fruits were pittango, mango and papaya while, in vegetables, the predominant types were carrot, dill, parsley, tomato, lettuce, sweet potato and red pepper. Red fruits and vegetables contained mainly lycopene. Yellow and orange fruits and vegetables had high contents of hydrocarbon carotenes with substantial levels of cryptoxanthins and xanthophylls. The green vegetables had high contents of both xanthophylls and hydrocarbon carotenes. Relatively high ratios (9- cis to all- trans β-carotene) of above 0.2 g/g were noted in sweet potato, papaya, parsley, lettuce, dill, apricot, pepper, prune and pumpkin, compared to the high ratio of 9- cis to all- trans β-carotene in the alga Dunaliella (~ 1.0 g/g). The high content of 9- cis β-carotene in certain fruits and vegetables and the wide variety of carotenoids and stereoisomers of carotenoids in all plants should shift nutritional and medical attention from the synthetic all- trans β-carotene toward natural carotenoids as potential candidates for chemoprevention.

New mode of Dunaliella biotechnology: two-phase growth for β-carotene production

Attempts to adapt the laboratory experience of bi-phasic growth and carotenogenesis in Dunaliella to large-scale conditions were highly successful. Algae were initially cultivated in stage one for optimizing biomass production of cells containing a low β-carotene to chlorophyll ratio. The culture was then transferred to stage two, diluted to about one third and induced for carotenogenesis. The bi-phase cultivation increased β- carotene productivity to 450 mg m-2 d-1 in stage one and to 300 mg m-2 d-1 in stage two, compared to the relatively low productivity of below 200 mg β- carotene m-2 d-1, in the conventional one phase type of cultivation. The results indicate that a selected algal product can be promoted specifically by growth manipulation of the alga and its environment.

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive β-carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of β-carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive β-carotene accumulation, greatly enhances β-carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive β-carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive β-carotene accumulation in D. bardawil.

Reduction of exercise-induced asthma oxidative stress by lycopene, a natural antioxidant.

Background: Lycopene has previously been shown to have high antioxidative activity. In view of the controversy regarding the beneficial effect of antioxidants on asthma, the acute effects of lycopene (LYC‐O‐MATOTM) on airway hyperreactivity were assessed in patients with exercise‐induced asthma (EIA).

Effect of Dietary Supplementation of Red or White Wine on Human Blood Chemistry, Hematology and Coagulation: Favorable Effect of Red Wine on Plasma High-Density Lipoprotein

Twenty healthy males were divided into two groups: 10 subjects were supplemented for 2 weeks with 400 ml of red wine (11% alcohol) per day and the other 10 subjects were given 400 ml of white wine (11% alcohol) per day for a similar period. Blood samples were drawn prior to wine supplementation, after 1 week and at the end of the study. No significant effects were found on plasma concentrations of urea, creatinine, bilirubin, creatine kinase, amylase, blood cell counts, platelet counts and platelet aggregation. Both red- and white-wine supplementation resulted in a transient minor reduction in plasma glucose concentration and in a minor elevation in blood coagulation properties such as prothrombin time and partial thromboplastin time. Red (but not white) wine resulted in an 11 and 26% increment in plasma triglyceride concentrations after 1 and 2 weeks of supplementation, respectively. Plasma cholesterol, as well as very-low- and low-density-lipoprotein levels did not change during the 2 weeks of red- or white-wine supplementation. The most impressive effect of red-wine intake was a significant (p < 0.01) increase in plasma high-density lipoprotein (HDL) cholesterol and in plasma apolipoprotein A-I concentrations by up to 26 and 12%, respectively. These effects were not observed after the intake of white wine. We conclude that the major effect of red-wine supplementation (about 40 g of alcohol per day for a period of 2 weeks) was a significant increase in plasma HDL concentration which may contribute to the reduced risk for cardiovascular diseases observed in red-wine drinkers.

Effect of a Mediterranean Meal on Postprandial Carotenoids, Paraoxonase Activity and C-Reactive Protein Levels

Background and Aim: Atherosclerosis involves oxidative and inflammatory mediators regulated by fat and antioxidants. Therefore, we studied the postprandial evolution of plasma lipids, carotenoids, C-reactive protein (CRP), and human serum paraoxanase activity (PON1) following two different fatty meals. Subjects and Methods: Eight healthy males consumed a 45% fat 1,000 Kcal Mediterranean-like (Med) meal (monounsaturated 61% of fat) compared to a Western-like (Wes) (saturated 57% of fat) meal. Blood was collected at baseline (time 0) 2, 4 and 7 h postprandial. Plasma lipids, glucose, insulin, total carotenoids, CRP, and PON1 were analyzed. Results: There was a marginal increase in cholesterol and glucose after both meals. Triglycerides increased modestly (to less than 200 mg/dl) and insulin increased (more in the Wes-like meal) but still within normal range, indicating a low glycemic index for both meals. Only the Med-like meal resulted in a significant increase in both PON1 activity (16%, p < 0.02) and carotenoids (74%, p < 0.02) with a 2-hour postprandial decrease in CRP (6%, p < 0.02). Conclusion: A postprandial monounsaturated fatty acid rich meal increases both plasma carotenoids and PON1 with a decrease in CRP levels, thus providing a novel potential explanation to the protective properties of a Mediterranean diet against atherogenesis.

Dietary Supplementation of a Natural Isomer Mixture of Beta-Carotene Inhibits Oxidation of LDL Derived from Patients with Diabetes mellitus

Background: Accelerated atherosclerosis is common in patients with diabetes mellitus which may be linked to increased lipid peroxidation. Therefore, we compared the oxidation of LDL derived from patients with diabetes to normoglycemic controls and followed-up the effect of dietary β-carotene supplementation on LDL oxidation. Methods: Twenty patients with long-standing non-insulin-dependent diabetes mellitus were studied in comparison with age- and sex-matched control subjects. Dunaliella bardawil-derived β-carotene was supplemented to the patients for 3 weeks, 60 mg daily dose. LDL oxidation was analyzed by measuring malondialdehyde (MDA), lipid peroxides (PD), and conjugated dienes (CD) generation in response to CuSO4-induced oxidation. LDL lipid composition and the LDL associated vitamins A, E and carotenoids were also measured. Results: LDL susceptibility to oxidation by CuSO4 was increased in the patients by 40% with a 35% shorter lag time required for the initiation of LDL oxidation, i.e. 56 ± 6 min in patients vs. 85 ± 9 min in controls (p <0.01). Patients showed increased cholesterol/phospholipid and polyunsaturated/saturated ratios, as well as reduced content of LDL associated vitamins. Upon β-carotene supplementation, there was a significant elevation in plasma and in LDL all-trans β-carotene [from 0.296 ± 0.020 to 0.968 ± 0.133 μg/mg LDL protein (p < 0.01)] paralleled by a significant reduction in LDL susceptibility to oxidation, as exhibited by increased lag time up to 115 ± 10 min (p < 0.01) and reduction in MDA and PD generation (by 25 and 40%), respectively (p < 0.01). Conclusions: Increased susceptibility to oxidation of LDL derived from patients with diabetes mellitus is associated with abnormal LDL lipid composition and antioxidant content. Natural β-carotene dietary supplementation normalizes the enhanced LDL oxidation and consequently may be of importance in delaying accelerated development of atherosclerosis in these patients.

Prevention of exercise-induced asthma by a natural isomer mixture of β-carotene

Background The unicellular alga Dunaliella bardawil was previously shown to contain very high concentrations of β-carotene composed of equal amounts of the all-trans and 9-cis stereoisomers which differ in their physicochemical features and antioxidative activity. Due to the controversy regarding the beneficial effect of antioxidants on asthma, the acute effects of β-carotene of Dunaliella was assessed on airway hyperreactivity in patients with exercise-induced asthma (EIA). Methods Thirty-eight patients with EIA participated in our study to verify the antioxidative effect. The test was based on the following sequence: baseline pulmonary function, 7 minutes exercise session on a motorized treadmill, 8 minutes rest, 1-week oral random, double-blind supplementation of placebo or 64 mg/day β-carotene, pulmonary functions at rest, 7 minutes exercise session, 8 minutes rest and again pulmonary functions. Results All patients given placebo showed a significant postexercise reduction of more than 15% in their forced expiratory volume in one second (FEV 1 ). Of the 38 patients who received a daily dose of 64 mg of β-carotene for 1 week, 20 (53%) were protected against EIA. Conclusions Our results indicate that a daily dose of Dunaliella β-carotene exerts a protective effect against EIA in some patients most probably through in vivo antioxidative effect.