AU596839B2

AU596839B2 - Use of D. bardawil as a source of vitamin A and a yolk-color enhancing agent

Info

Publication number: AU596839B2
Application number: AU71864/87A
Authority: AU
Inventors: Mordhay Avron; Ami Ben-Amotz; Samuel Edelstein
Original assignee: Yeda Research and Development Co Ltd
Current assignee: Yeda Research and Development Co Ltd
Priority date: 1986-04-23
Filing date: 1987-04-22
Publication date: 1990-05-17
Anticipated expiration: 2007-04-22
Also published as: GB2189675B; GB8709638D0; AU7186487A; GB2189675A; IL78608A0

Description

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COMMONWEALTH 'F AUSTRALIA Patent Act 1952 596839 COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class ligk4i~7 Application Number Lodged Complete Specification Lodged Accepted Published Thi documet conto l atedmclts made under S:ctil n 49 and is correct for 23 April 1986 "'Priority: SRelated Art Qo 4 Name of Applicant YEDA RESEARCH AND DEVELOPMENT COMPANY

LIMITED

P.O. Box 95, Rehovot, Israel .'Address of Applicant Actual Inventor Address for Service 0 a Mordhay Avron, Ami Ben-Amotz, Shmuel Edelstein F.B. RICE CO., Patent Attorneys, 28A Montague Street, BALMAIN. 2041.

Complete Specification for the invention entitled: USE OF D. BARDAWIL AS A SOURCE OF VITAMIN A AND A YOLK-COLOR ENHANCING AGENT The following statement is a full description of this invention including the best method of performing it known to us:- FIELD OF THE INVENTION: There is provided a feed supplement for poultry which serves both as source of vitamin A and enhances the yellow color of the egg yolk. The supplement is based on carotene-rich algae of the genus Dunaliella.

BACKGROUND OF THE INVENTION: In US Patents No. 4,115,949, 4,190,895 and Plant patent 4,511 there is described the cultivation of Dunaliella algae as a source of glycerol beta-carotene and proteinous substances of nutritive value. It is known that certain oxy-carotenoids such as lutein when added to chicken-feed enhance the yellow color of egg yolks, which is the preferred color in many markets.

o o. SUMMARY OF THE INVENTION: t Some algae of the genus Dunaliella, and especially Dunaliella Sbardawil, (see US Plant Patent No. 4,511) can be used as source of vitamin A and also as agent enhancing the yellow coloration of egg yolks when administered to poultry.

As source of vitamin A there may be used lyopnylized algae, wet o a o. algae or algae dried by other means such as drum or spray drying.

The algae are cultivated as set out in said US patents or by any i other conventional culture process, under conditions which lead to enhanced beta-carotene content (Ben-Amotz and Avron: On the Factors which determine massive beta-Carotene Accumulation in the "o Halotolerant Alga Dunaliella bardawil, Plant Physiol. vol. 72, 593-597 (1983).

a l ""g.C~1~"4nrYPs~ L3 -ICIC- rra-e~lr The quantities of feed supplement to chicken feed in or ler to supply the required quantity of vitamin A is from about 0.1 to about 1 g per 1000 g chicken feed. The quantity for enhancing the yellow color of the egg yolks is from about 1 g to 10 g per 1000 g animal food.

Figures 1-3, set out the use of Dunaliella bardawil as a source of vitamin A in chick diet: mean body weight against time from initial day of growth.

The following experiments were conducted to demonstrate the use of algae of the Dunaliella species as vitamin A source and as agent 10 enhancing the yellow coloration of egg yolks: ooo S 0 0 o o 0 0 o 00 a I o0 0t 0, 0 f s vf Example 1 was conducted with 15 vitamin A deficient chicks divided into three groups (at the stage where they showed obvious vitamin A deficiency symptoms): 1) Five chicks continued to receive the vitamin A deficient diet.

2) Five chicks received a vitamin A supplemented diet.

3) Five chicks received the vitamin A deficient diet supplemented with lyophylized D. bardawil (containing salt and 6% beta,-carotene) at a level of 4 g/kg feed.

The results were: 1. In terms of growth and general appearance groups 2 and 3 were similar and markedly superior to group 1. The differences were evident already after 2 days. Two of the chicks in group 1 died at the end of the 7 day feeding period.

2. Blood analysis by HPLC of the chicks after 7 days feeding indicated that group 2 and 3 contained normal levels of vitamin A in their serum. No vitamin A could be detected in the serum of the surviving chicks in group 1.

Example 2 was conducted with 6 egg-laying young chickens divided into two groups (within a commercial egg-producing farm): 1. Three chickens received their normal diet (containing 150 g/Kg Corn-meal which is a source of carotenoids) 2. Three chickens received the same diet supplemented with 4 g/kg of lyophylized algae (containing 30% salt and beta-carotene.

The egg-yolks of both groups were examined daily visually for their yolk-color, and spectrally in an acetone extract. No difference was 1 0 detected during the first two days. However, eggs layed following three days feeding were learly a deeper yellow in group 2, and the difference in color maximized after the 5th day. IHPLC analysis showed that the o yolk color was due to lutein, and not to beta-carotene, and that the lutein content from the maize meal in the diet was increased by the D.

ot bardawil supplement. The color difference was maintained throughout the 30 day experimental period.

o 0 a o O oss o o o O0 0 0 9 Example 3 was conducted with 40 one-d-old White Leghorn chicks divided into 4 equal groups. All received a vitamin A-deficient semi-purified diet or diets supplemented with 8 mg vitamin A/kg food, synthetic beta-carotene at 30 mg beta-carotene/kg food, or lyophylized D. bardawil at 1 g algae/kg food. The lyophylized D. bardawil contained 3% beta-carotene and 20% NaCl (30 mg beta-carotene/kg food).

Example 4 was conducted with 40 one-d-old chicks divided into 4 equal groups. All received the vitamin A-deficient diet or diets 3supplemented with 8 mg vitamin A/kg diet, synthetic beta-carotene at mg beta-carotene/kg or drum dried D. bardawil at 1 g algae/kg diet. The drum dried D. bardawil contained 3.4% beta-carotene, 26% NaC1, 18% glycerol, 0.6% chlorophyll, 21% protein, 17% carbohydrate and 12% lipid (34 mg beta-carotene/kg diet).

Example 5 was conducted with 45 one-d-old chicks divided into three equal groups. All received a vitamin A-deficient diet or that diet supplemented with lyophylized D. bardawil it 0.58 g algae/kg diet, or drum dried D. bardawil at 1 g/kg diet. The lyophylized D. bardawil i 10 contained 5.5% beta-carotene (32 mg beta-carotene/kg diet). The drum dried D. bardawil was the same as that described in experiment 4 (34 mg beta-carotene/kg diet).

o 0 OS Example 6 was conducted with 160 one-d-old chicks, divided into 4 0 00 o i' equal groups. All received a vitamin A deficient diet or that diet supplemented with lyophylized D. bardawil at 0.5 g or 2.50 g algae/kg 6 0 o a feed, or supplemented with synthetic beta-carotene at 25 mg or 125 mg/kg feed. The lyophylized D. bardawil contained 5% beta-carotene o 0 o 0 o The chicks in experiments 3-6 were assessed visually and weighed S*o daily individually during the growth period, which lasted for 5 weeks in each experiment.

Vitamin A, beta-carotene and lutein in the serum and the liver o0, were extracted. Chick serum (0.5 mi) or liver (0.5 g) were extracted in water:cthanol:petroleum ether (1:1:5 v/v) under N 2 After phase separation, the upper phase was divided into two equal volumes and 4i evaporated to dryness. One was dissolved in a minimal volume of methanol:water (9:1 v/v) for vitamin A determination by IIPLC and the other was dissolved in a small volume of methylene chloride for determination of beta-carotene and lutein by HPLC.

The significance of all treatment differences was determined by the t-test.

Fig. 1 and 2 illustrates the weight gain of the chicks in experiments 3-5, and Fig. 3 in experiment 6. Each included a control group of chicks fed on a vitamin A-deficient diet. Within 20 d the 10 vitamin A-deficient chicks showed obvious loss of weight, and general weakness. Some of the chicks on the vitamin A-deficient diet died during the 5 weeks of each experiment, but no significant mortality was o0 S observed in the other groups. The two other control groups included oo chicks fed on a vitamin A-deficient diet supplemented with vitamin A So or beta-carotene. These chicks were normal both in appearance and o 0 o weight gain. Within 4 to 5 weeks their weight increased about 10 fold.

Similar growth patterns and weight gains were observed whether the vitamin A was added to the food or was injected subcutaneously (not I shown).

i O The potential of D. bardawil to supply the vitamin A .o requirement of chicks was determined with either lyophylized or drum-dried algae. As can be seen in Fig. 1 and 2 chicks fed on diets o supplemented with vitamin A, beta-carotene, lyophylized or drum-dried algae containing an equivalent beta-carotene content showed similar weight gains. In appearance, the chicks fed on the algae-supplemented diet looked slightly better with more intensive pigmentation.

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0 0 Figure 3 illustrates the average growth of larger groups of chicks in each group, example 6) fed synthetic beta-carotene or lyophylized D. bardawil containing the same amount of beta-carotene. The numbers in parentheses indicate the surviving chicks in the group fed with the vitamin A deficient diet. As can be seen no significant difference was observed between chicks fed with equal amounts of beta-carotene in the form of a synthetic material or in D. bardawil. It may be noted that the group fed with 125 mg synthetic beta-carotene/kg diet grew somewhat slower that that fed with 25 mg synthetic beta-carotene/kg diet, but there was no difference in the growth rate of the chicks fed with 0.5 g or 2.5 g D. bardawil/kg diet (not shown in figure).

Blood and liver analyses for vitamin A, lutein and beta-carotene content are summarized in the Table. No vitamin A was detected in the sera or livers of the surviving chicks of the vitamin A-deficient group. The sera of chicks fed vitamin A, synthetic beta-carotene or D.

bardawil contained adequate amounts of vitamin A. The livers of the chicks fed with beta-carotene and D. bardawil contained vitamin A and vitamin A esters (mostly vitamin A-palmitate) in a ratio of 1:4 and in amounts which increased with the increase in the amount in the diet.

Previous attempts to supply the vitamin A requirement in animal feed by algal supplementation failed because: Most algae are enclosed by a cellulotic cell wall which is indigestible by chickens and most other animals, including humans, and Most algae contain relatively small amounts of beta-carotene (less than The halotolerant alga Dunaliella bardawil is unique in containing no indigestible cellulotic cell wall and in its ability to accumulate a high content of beta-carotene, in excess of 100 g/kg of algal dry weight.

6- The dried algae of this type, rich in beta-carotene is a highly efficient source of vitamin A in the normal diet of poultry and enhances the yolk color of eggs in laying hlens.

It was found that 0.5 g of dried algae per kg of vitamin A-deficient diet was more than sufficient to provide the complete vitamin A requirements of chicks. The beta-carotene in dried algae is stable over prolonged periods of time.

aco *9i o 09 0 9 t *00*l S0 I o a i 099919 o o t 0 0* 0 9* Sao 0 0 o (a 0 9 0 0 0 0 rttJ 4 -7- Vitamin A, Vitamin A palmitate, lutein and beta-carotene in the serum and liver of chicks fed on a vitamin A deficient diet or supplemented A, or adequate and excessive levels of beta-carotene in the form of synthetic material or dry Dunaliella bardawil.

Serum Liver Vitamin A Luteina Vitamin A Vitamin A beta-carotene palmitateb all-trans 9-cis -mr t 4& ;NcVitamin A Vitamin A 8 mg/kg diet Synthetic beta-carotene 25mg/Kg diet "125 mg/Kg diet Dry Dunaliella bardawil 0 0.5g/Kg diet f5mg beta-carotene) diet (125 mg beta-carotene) Ig x mr' jg x gr.fresh weight- 1 0.0 26.4 36.2 23.6 66.6 >0.1 0.3 1.7 16.6 aMostly lutein but also unidentified xanthophyll.

bMostly vitamin A-palmitate with minor content of other vitamin A esters. No vitamin A esters were found in the serum.

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Claims

1. A feed supplement, to provide a source of vitamin A and to enhance the yellow colour of eggs when administered to laying hens, when used i. 4 animal feedstuff comprising an effective quantity of beta-carotene rich algae of the Dunaliella genus and a suitable feed base.

2. A supplement according to claim 1, wherein the algae are Dunaliella bardawil.

3. A supplement according to claim 1 or 2 in the for= cf lyophylized or otherwise dried algae.

4. A supplement according to any of claims 1 to 3, wherein the quantity of algae added to the poultry *.to feedstuff is from about 0.1 g to about 4 g of alcae per 1000 g feedstuff.

5. A process for augmenting the yellow color of egg 4 4 yolks which comprises administering to poultry at least one gram of algae of the Dunaliella genus per one tho.nusa.d grams of animal food. DATED this 9th day of January 1990 YEDA RESEARCH AND DEVELOPMENT COMPANY 1'_-D Patent Attorneys :or the Applicant: F.B. RICE CO. *a