RbodospiriUnm and Phaeospirillum in animal feed for the purpose of obtaining food products with a reduced cholesterol level

The invention relates to the use of Rhodospirillum spp. and/or Phaeospirillum spp. as animal feed for production animals for the purpose of decreasing the cholesterol level of food products obtained from the production animals. The invention also relates to a method for obtaining foods with a decreased cholesterol level. The invention also relates to a food with a decreased cholesterol level. The invention further relates to an animal feed for production animals.

A high cholesterol level in the blood is seen as a factor which increases the risk of cardiovascular disease in people. A significant factor in obtaining a high cholesterol level is the intake of cholesterol from food. It is desirable to consume food with a low cholesterol level. It is however difficult to bring about a low cholesterol level in natural products from production animals (including cows, pigs and chickens), such as meat and milk. This is also the case for derivative products such as butter.

The invention has for its object to provide a simple method of decreasing the cholesterol level of products of animal origin.

The invention provides for this purpose the use of Rhodospirillum spp. and/or Phaeospirillum spp. as animal feed for production animals for the purpose of decreasing the cholesterol level of food products obtained from the production animals. Rhodospirillum spp. and Phaeospirillum spp. are bacterial strains which, when used continuously as feed for animals, are found to reduce the cholesterol level of products obtained from these animals when compared to animals having the same diet without Rhodospirillum spp. and/or Phaeospirillum spp. Rhodospirillum spp. and Phaeospirillum spp. can be used as food in living form or as preparation obtained from the bacteria, such as a freeze-dried preparation or an extract. One species of bacteria can be used, although it is also possible to envisage mixing and feeding different genera of bacteria as mixture to the production animals. Production animals include for instance: mammals, in particular ruminants such as cows and sheep; poultry, in particular chickens and turkeys; and cultured fish, in particular salmon, trout and perch. The

bacteria are easy to culture and process according to known methods, and they can also be readily incorporated into different types of animal feed.

Rhodospirillum is a genus of the family Rhodospirillaceae, a family of purple, non- sulphur bacteria. Three species are distinguished within the genus Rhodospirillum: Rhodospirillum rubrum (Imhoff and Truper, 1992), Rhodospirillumcenteπum and Rhodospirillumphotometricum. Also known are four subspecies which are not officially recognized: Rhodospirillumsalexigens, Rhodospirillum salimarum, Rhodospirillum sodomense and Rhodospirillum tenue. Included within the genus Phaeospirillum, which is likewise part of the family Rhodospirillaceae, are two species: Phaeospirillum fulvum and Phaeospirillum molischianum. For purposes of nomenclature reference is made to Imhoff JF, Petri R, Suling J, Int. J. System . Bacteriol. 1998, 48, 793-798 and Deutsche Sammhing von Mikroorganismen und Zellkulturen GmbH. Bacterial nomenclature: www.dsmz.de/bactnom/bactname.htm.

It is known from WO 2004/052380 that genera of Rhodospirillum and Phaeospirillum can lower the cholesterol level in blood and plasma of special mouse models. Surprisingly, it is also found possible using the same bacteria to reduce the cholesterol level in the fat and meat of production animals, as well as in milk and eggs obtained from these production animals. Production animals are understood to mean animals (including cows, pigs, chickens and turkeys) which provide products (meat, fat, eggs, milk) which are suitable for human consumption; test animals such as the special mouse types described in WO 2004/052380 are not included here.

The genera of Rhodospirillum en Phaeospirillum described in the international application WO 2004/052380 (inventors Emeis and Lasseur) are found to be particularly suitable as food for production animals for the purpose of decreasing the cholesterol level in products from these production animals.

In a preferred embodiment the production animal is a mammal, wherein the food product comprises meat products and/or dairy products. The dairy products comprise milk and products derived therefrom such as butter, yoghurt and cheese. Particularly suitable mammals are cattle, pigs, sheep and goats.

In another preferred embodiment the production animal is poultry, wherein the food product comprises meat products and/or eggs. Particularly suitable types of poultry are chickens, turkeys, ostriches and ducks.

Rhodospirillum spp. as used according to the invention preferably comprises at least one strain selected from the group consisting of Rhodospirillum rubrum, Rhodospirillumcentenum, Rhodospirillumphotometricum, Rhodospirillumsalexigens, Rhodospirillum salimarum, Rhodospirillum sodomense and Rhodospirillum tenue.

Phaeospirillum spp. preferably comprises at least one strain selected from Phaeospirillum fiilvum or Phaeospirillum molischianum.

The invention also provides a method for obtaining foods with a decreased cholesterol level, comprising the processing steps of: feeding a production animal with an animal feed comprising Rhodospirillum spp. and/or Phaeospirillum spp. in a quantity and for a period of time such that the cholesterol level of an intended food product obtainable from the production animal is reduced compared to the cholesterol level of a production animal fed with an animal feed without Rhodospirillum spp., and obtaining the food product with decreased cholesterol level from the production animal. This method makes it possible in simple manner to obtain food products with a decreased cholesterol level compared to products from animals on a diet without Rhodospirillum spp. and/or Phaeospirillum spp. A content of 0.5% by weight of Rhodospirilhun spp. and/or Phaeospirillum spp. in the supplied feed, administered over a period of at least two weeks, is generally sufficient to measure a significant decrease in cholesterol in the obtained products. Preferably however, administering takes place over a continuous period of at least four weeks. The content of Rhodospirillum spp. and/or Phaeospirillum spp. is preferably at least 1% by weight, whereby a lower cholesterol level is realized in the products, and the desired level is reached more quickly. Levels higher than 15% by weight of Rhodospirillum spp. and/or Phaeospirillum spp. result in practice in only a minimal further decrease, compared to 15% by weight, in the cholesterol levels measured in the products. Rhodospirillum spp. and/or Phaeospirillum spp. can be administered in living form or as preparation. The stated levels are for living bacteria; an equivalent quantity of preparation can likewise be used.

The invention further provides a food product with a decreased cholesterol level obtainable by means of the method according to the invention. Particularly included here are meat products and fat products obtained from slaughtering animals fed with the method according to the invention. Products such as eggs, milk and products derived therefrom are also included here.

The invention also provides an animal feed for production animals, comprising Rhodospirillum spp. and/or Phaeospirillum spp. Such an animal feed makes it readily possible to reduce the cholesterol level of products from production animals fed with the animal feed according to the invention.

The animal feed preferably comprises at least 0.5% by weight of Rhodospirillum spp. and/or Phaeospirillum spp. When feeding takes place with such an animal feed, a significant decrease in the cholesterol level in the obtained products is measured within a few weeks.

In a preferred embodiment the animal feed comprises between 0.5 and 15% by weight of Rhodospirillum spp. and/or Phaeospirillum spp. Above 15% by weight the additional cholesterol-reducing effect in products, compared to 15% by weight, is minimal and therefore not a very efficient use of the relatively expensive Rhodospirillum spp. and/or Phaeospirillum spp.

The animal feed more preferably comprises between 0.5 and 5% by weight of Rhodospirillum spp. and/or Phaeospirillum spp. At such a percentage a decreased cholesterol level in the products is achieved in simple manner and without side-effects within a period of a few weeks. Between 5 and 15% by weight the additional cholesterol-decreasing effect, compared to 5% by weight, is relatively small and therefore less efficient.

In a preferred embodiment the animal feed is suitable for mammals, comprising between 0.5 and 15% by weight of Rhodospirillum spp. and/or Phaeospirillum spp. supplemented with mammal feed. Mammal feed can comprise both fodder and concentrated feed, wherein fodder generally comprises fresh grass, grass silage and/or maize silage, and the concentrated feed consists of extra added nutrients, in particular

proteins, vitamins and minerals. Any suitable commercially available feed can in principle be used. Rhodospirillum spp. and/or Phaeospirillum spp. are preferably mixed with the concentrated feed, since this is most easily dispensed. The precise composition is related to, among other factors, the type of mammal and the intended result, for instance meat production or milk production.

In another preferred embodiment the animal feed is suitable for poultry, comprising between 0.5 and 15% by weight of Rhodospirillum spp. and/or Phaeospirillum spp. supplemented with poultry feed. Poultry feed is a mixture of fodder and concentrated feed, wherein fodder consists particularly of seeds such as maize, grain and/or linseed, and the concentrated feed consists of extra added substances, in particular proteins, vitamins and minerals. Any suitable commercially available feed can in principle be used. Rhodospirillum spp. and/or Phaeospirillum spp. are preferably mixed with the concentrated feedj since this is most easily dispensed. The precise composition is related to, among other factors, the type of mammal and the intended result, for instance an improved meat production or egg production.

In yet another preferred embodiment the animal feed is suitable for fish, comprising between 0.5 and 15% by weight of Rhodospirillum spp. and/or Phaeospirillum spp. supplemented with fish feed. Fish feed generally comprises in particular proteins and fats of animal origin, optionally supplemented with concentrated feed comprising extra proteins, vitamins and minerals. Any suitable commercially available feed can in principle be used. The precise composition is related to, among other factors, the type of mammal and the intended result.

The invention will now be elucidated on the basis of the following examples.

Example 1: Production of low-cholesterol dairy products

Ruminants for milk production (cows, sheep and goats) were fed with a mixed feed comprising fodder and concentrated feed comprising all essential nutrients. Further added to this concentrated feed was 0.5-5% (by weight) R. Rubrum. From 6 to 12 weeks after the beginning of this feeding (depending on the dosage, breed of the animal and composition of the other feed) a significant reduction in the cholesterol level was measured in the milk fat. When the milk was processed into dairy products, this

decreased cholesterol level likewise became manifest; the reduction in cholesterol level was also measured in the milk, butter, cheese and other dairy products prepared with this milk. Used in this example was Rhodospirillum rubrum which gave the best results and has a good processability; good results can however also be achieved with the other bacteria according to the invention. In addition to the usual fodder (fresh grass and/or grass/maize silage), the animals received concentrated feed comprising minerals, vitamins and extra proteins mixed with Rhodospirillum rubrum as according to Table 1. Mixture 2, with a higher dosage, also showed a greater decrease in the cholesterol level of the produced milk than mixture 1. Mixture 3 gave results not differing much from mixture 2.

Table 1: Composition of feed (percent by weight)

Example 2: Production of low-cholesterol meat and meat products

Agricultural animals kept for meat production (pigs, beef cattle, fish and poultry) were fed with a feed comprising all essential nutrients for the relevant animal species. Further added to this feed was 0.5-5% (by weight) R. Rubrum; the compositions used are shown in Table 2. 4-12 weeks after the beginning of this feeding a significant reduction in the edible parts (meat, organs and fat) of the slaughtered animal was measured using known measuring methods; this reduction was also measured in meat products produced with these parts. Mixture 4 contained only Rhodospirillum rubrum, Mixture 5 both Rhodospirillum rubrum and Phaeospirillum fulvum, and Mixture 6 only Phaeospirillum fulvum. AH three mixtures gave comparable results in cholesterol reduction in the produced meat.

Table 2. Composition of feed (percent by weight):

Example 3: Production of low-cholesterol eggs

Laying hens were fed with a feed comprising all essential nutrients. Further added to this feed was 0.5-5% (by weight) R. Rubrum as shown in Table 3. 8-18 weeks after the beginning of this feeding a significant reduction in the cholesterol level in the yoke of the egg was measured; this reduction was also measured in egg products produced with these eggs. Mixture 8 used a relatively high dosage of Rhodospirillum rubrum, and showed a significant decrease in the cholesterol level relative to mixture 7.

Table 3: composition of feed (percent by weight):

Example 4: Low-cholesterol eggs

In this test the cholesterol level of consumer eggs was reduced by adding Rhodospirillum rubrum in a composition according to the invention to laying hen feed for a period of 4 weeks.

Both brown and white laying hens were used for this test because of possible differences in their fat and protein metabolism. In addition to the cholesterol level of the eggs, an analysis of cholesterol and triglycerides in the blood was also done at the end of the test period.

Test setup

Animals and accommodation

o

Two different breeds of laying hen were used for this test: brown and white, because of possible differences in their fat and energy metabolism. The laying hens had an age of 30 weeks. The laying hens were housed per 2 in a section of a laying battery in order to thus obtain a precise overview of production characteristics such as laying percentage, feed intake and chicken and egg weights. The laying hens had a dark period of 6 hours.

Test 2τoups

This test consisted of two different test groups:

Group A = control group: laying hens received standard maize free-range feed containing about 5% fat. Number of brown chickens: 7 Number of white chickens: 6

Group B = Rhodospirillum rubrum group: laying hens receive the same standard maize free-range feed as group A, but with the addition of 5% Rhodospirillum rubrum.

Number of brown chickens: 2

Number of brown chickens: 2

The composition of both feeds is shown in Table 7.

The laying hens were given limited feed (125 g/d) because they would use less energy due to the accommodation of free-range chickens in a laying battery arrangement. Water was available ad libitum.

Table 7: Composition of the test feeds (%). Ingredient Control feed Rhodospirillum rubrum feed

Maize 62.75 63.02 Soya hipro 20 14.73 Limestone pellets 7.5 7.5 Rhodospirillum rubrum 5

Zonblosr RC 200-240 4.03 4.36

Palm oil 2.34 2.02

Chalk 1.97 1.99

Premix 0.5% std 0.5000 0.5000

Monocalcium phosphate 0.4497 0.4354

Salt 0.2795 0.2716

DL-methionine 0.1308 0.1423

L-tryptophan 98.5% - 0.0024

Vitamin E 50 ADS 0.0200 0.0200

Natuphos 500 FTU/g 0.0060 0.0060

Sodium selenite 5% DF 0.0006 0.0006

Calculated nutrients, g/kg

Crude protein 160 160

Crude fat 51.7 49.0

Crude fibre 30.0 30.0

Starch 405 408

Ca 40.0 40.0

P 4.4 4.7

ME, kcal 2850 2850

Measurements of technical performance

The laying hens were weighed on days 1, 15 and 29. The laying percentage was recorded daily. The eggs were collected weekly (days 1, 8, 15, 22 and 29) for the purpose of determining the weight. On days 0, 14 and 28 the eggs were collected for the purpose of a standard entry check (weight, egg white height, Haugh unit and Roche colour), which was performed by Natuurfarm De Boed/WEKO, Ochten, the Netherlands.

Blood samples for the purpose of cholesterol and triglyceride levels were also taken once-only at the end of the test period. These blood samples were analyzed by the University Veterinary Diagnostic Laboratory, Utrecht, the Netherlands.

Results

The results of the weights of the laying hens, the feed intake and growth are shown in Table 8.

Table 8 : Average body weight, food intake and growth speed of the brown and white laying hem for four weeks for each feed group

Control group Rhodospirillum rubrum group

Brown laying hens Weight, kg Day l 1.87 2.39 Day 15 1.96 2.55 Day 29 2.05 2.61

Food intake, g/d 124 125 Growth, g/d 6.4 7.9

White laying hens Weight, kg Day l 1.64 1.41 Day 15 1.70 1.48 Day 29 1.81 1.55

Food intake, g/d 117 115 Growth, g/d 6.2 5.0

Table 6 shows the weights of the eggs and the laying percentage is during the test period for both test groups. For both the brown and the white eggs it is the case that the weights fluctuate somewhat between the different measuring times. The laying percentage of the white chickens in the control group is on the low side.

Table 6: Average egg -weight and laying percentage of the brown and white laying hens for four weeks for each feed group.

Control group RJiodospirillum rubrum -group

Brown laying hens Egg weight, g Day O 59.7 67.3 Day 14 61.3 61.7 Day 28 60.5 62.4

Laying percentage, % Day 1-29 99.4 95.0

White laying hens Egg weight, g Day O 58.4 59.5 Day 14 61.7 62.3 Day 28 60.9 55.4

Laying percentage, % Day 1-29 80.6 96.7

The data for the yoke colour (Roche scale) are given in Table 7. After two weeks the laying hens given Rhodospirillum rubrum in their feed had a considerably higher Roche colour (darker yoke) than the laying hens of the control group. The Rhodospirillum rubrum feed according to the invention is much redder than the control feed; Rhodospirillum rubrum evidently also has a secondary effect as a yoke-colouring agent.

Table 7: Average yoke colour (Roche-scale) of the brown and white laying hens for four weeks for each feed group

Control group Rhodospirillum rubrum -group

Brown laying hens

Yoke colour DayO 4.0 4.0 Day 14 6.6 11.0 Day 28 6.4 11.0

White laying hens Yoke colour Day O 2.6 2.5 Day 14 6.6 10.5 Day 28 6.6 11.0

Cholesterol and triglyceride analyses

At the end of the test blood was also taken from two laying hens from each group, from the wing, in order to thus gain an impression of blood parameters, i.e. overall cholesterol and triglycerides. The cholesterol and triglyceride level forms a measure of the level of LDL cholesterol in the eggs. The results of these analyses are shown in Table 8. It can be concluded herefrom that both the cholesterol and the triglyceride level are reduced as a result of Rhodospirillum rubrum in the feed of laying hens.

Table 8: Overall cholesterol and triglycerides (mmol/l) in blood of the brown and ■white laying hens at the end of the pilot test

Control group Rhodospirillum rubrum -group Brown laying hens

Cholesterol 4.8 2.3

Triglycerides 30.5 13.1

White laying hens

Cholesterol 5.4 2.0

Triglycerides 32.6 10.6

Conclusion

Use was made in this test of two different breeds because the literature has shown that the fat and energy metabolism can differ between these breeds. Production characteristics such as body weight, food intake, egg weight and laying percentage are based on small numbers of animals, but do not differ essentially between the two groups. The cholesterol and triglyceride level in the blood of both white and brown chickens is reduced as a consequence of adding Rhodospirillum rubrum.