WO1998031377A1

WO1998031377A1 - Use of food products for protection against radicals

Info

Publication number: WO1998031377A1
Application number: PCT/NL1998/000034
Authority: WO
Inventors: Mohammed Essaidi; Frederik Albert Huf
Original assignee: Mohammed Essaidi; Frederik Albert Huf
Priority date: 1997-01-20
Filing date: 1998-01-20
Publication date: 1998-07-23

Abstract

The invention relates to the use of whey and products released from microbial fermentation, for preparing a foodstuff or medicament for reducing the effect of radicals in humans or animals, in ageing and diseases and the side-effects of radiotherapy, chemotherapy or AIDS combination therapy, for intensive care or the treatment of post-operational conditions, in the treatment or prophylaxis of leaky intestine, (auto)immune diseases, infections including viruses or prions, diabetes, migraine, neurodegenerative diseases, cancer and cardiovascular diseases, or for the treatment of stress conditions or excessive exercise, as well as to the corresponding food and drug products.

Description

Use of food products for protection against radicals

Background

Every living creature is continuously producing radicals because of its own oxidative metabolism. Those radicals however are immediately removed by natural processes such as recombination and enzymatic removal (for example superoxide dismutase

(SOD), catalase and glutathione-peroxidase). Radicals are formed in larger concentrations under more excessive circumstances such as sport, stress, hypoxemia as in ischaemia followed by reperfusion, radiotherapy, chemotherapy, infection and inflammation, but also accompany or cause illnesses like diabetes, cancer, cardio- vascular disease, arthritis, neurodegenerative diseases, (auto)immune diseases and other diseases. More generally, radicals are regarded to be an important cause of ageing processes. In case of (auto)immune diseases, a connection with the increased permeability of the intestine can be made, caused by stress, physical strain, hypoxemia/reperfusion, alcohol, food metabolites, drugs, xenobiotics, aminoacid deficiency, infections and inflammation and therefore enabling translocation from the lumen of toxins, prions, viruses and micro-organisms or their fragments present in the intestine and the immuno-response of the host. In all above cases the normal radical removing system is thought not be fast enough to remove radicals in time. The remaining radicals can cause damage to e.g. DNA causing fast mutations, eventually leading to cancer, and can also result in other diseases as indicated above. Also in the ordinary metabolism it is assumed that upon growing older the removing system slows down, resulting in radical concentrations which can again cause damage.

The body is normally assisted in its struggle to keep the radical concentration within the body negligible, by natural compounds taken in with food and/or formed as metabolites. There are several compounds and metabolites which react with radicals or radical forming compounds, such as for example vitamins (E and C), carotenoids, flavonoids and polyphenols (B. Halliwall and J.M.C. Cutteridge, "Free Radicals in Biology and Medicine", Clarendon Press, Oxford, 1996, p. 86-188 and p.416-494; ILSI Monograph, "Oxidants, Antioxidants and Disease Prevention", ILSI Press, Brussles, 1995, p. 13-23).

Particularly older people and people with unhealthy eating habits or lifestyle (for example alcoholics, smokers, drug users etc.) can have problems with respect to this basic amount of scavengers present, the lack of which can move those people into a higher risk category, particularly with respect to cancer, cardiovascular and neurodegenerative diseases and (auto)immune diseases. Similar radical mediated diseases can occur in animals.

Foodstuffs known to contain substantial amounts of different types of scavenging molecules include soybeans, tea, coffee, red wine, rosemary, sage and other spices, citrus and other fruits, onions, olives, tomatoes, carrots, broccoli etc. In some cases combinations of food products may enhance the radical scavenging.

Most of the scientific work underlying the radical scavenging is based on knowledge of the chemical properties, such as the redox potential of the different molecules. Little is known about the efficacy of the different compounds particularly with respect to the availability in the body and the reaction time under e.g. conditions in blood and organs.

Description of the invention

The invention pertains to the use of a combination of products which is effective in preventing the formation of radicals in the human and animal body and/or in scavenging such radicals. The invention also provides a method which allows the measurement of radical levels both in vivo and in vitro.

In particular, the invention relates to the combined use of whey solids and products released through fermentation by microorganisms, for preparing a foodstuff or medicament for the protection of the human or animal body against the effect of radicals. The protection against the effect of radicals applies especially to ageing and diseases and side-effects of radiotherapy or chemotherapy, combination therapy in the treatment of AIDS, to intensive care or the treatment of post-operational conditions, in the treatment or prophylaxis of diabetes, migraine, neurodegenerative diseases, cancer and cardiovascular diseases, or to the treatment of stress conditions, slimming or excessive exercise as well as (auto)immune diseases caused by pathogenic agents as toxins, prions, micro-organism or their fragments, which can be translocated from the lumen because of a radical-mediated increase of permeability of the intestine, caused e.g. by infections, drugs, xenobiotics, alcohol, aminoacid deficiency, hypoxemia as in ischemia followed by reperfusion, stress or physical strain. Furthermore the conversion is improved in animal feeds. Less antibiotics are needed in animal farming.

The mechanism of the protective effect of the method of the invention is believed to be multiple. Firstly, the formation of radicals is decreased by the active components. The decomposition of water by radiolysis is increased by hydrogen peroxide, and the removal of hydrogen peroxide by enzymes such as peroxidases and catalases decreases the damage. Also the damage from superoxide ions resulting from the presence of oxygen and superoxide dismutase is decreased. Furthermore there is extinction of radiation by iron compounds. Superoxide anion and hydrogen peroxide production resulting from hypoxemia followed by reperfusion is decreased by enzymes supplied by the fermentation products. Production of radicals during immunoresponse is not decreased as there is less formation of fatty acid oxidation products, since the body cells are protected against membrane destruction (J.J. Dibner et al, Animal Feed Sc. Tech. 62 (1996) 1-13).

Secondly, there is inactivation of radicals by microbial enzymes such as SOD, catalase and peroxidase. Thirdly, there is competitive scavenging during radical reactions by decrease of second order reactions of radicals with critical biomolecules as antioxidants and aromatic compounds competitively react with radicals. Fourthly, the general level of oxidation is decreased, and finally the presence of antioxidants, e.g. thiols, prevents further oxidation of damaged biomolecules and will protect or even restore these.

The whey solids preferably comprise whey proteins. Whey may be used as such, or in a concentrated form or in a low-lactose form. An effect is already obtained at a daily dosage of about 100 mg, but the preferred level of whey solids, especially whey proteins, is from 200 mg to 60 g, in particular 1-30 g of whey solids per day.

The products released through fermentation by microorganisms comprise enzymes and other metabolic products or parts of the microorganisms. The microorganisms may be bacteria, yeast and fungi, preferably food-grade. In particular, the microorganisms are selected from lactic acid bacteria and yeasts. Suitable lactic acid bacteria include the genera Lactobacillus, Lactococc s, Streptococcus, Pediococcus, Vagococcus, Leuconostoc, Carnobacterium, Weisselia, Oenococcus, Lactosphaera, Micrococcus, Bifidobacterium and Propionibacterium. Bacteria that can tolerate X-ray, gamma, beta and other radiation, such as Micrococcus radiodurans and Arhtrobacter tolerans are also suitable. Suitable yeasts include the genera Saccharomyces (ellipsoides and other) and Kluyveromyces . The microorganisms themselves may be present (preferably at least 10⁶ cells per daily dosage up to about 10¹⁰ cells per day) or their parts thereof or cell excretion products only. Active enzymes include oxidoreductases such as SOD, catalase, peroxidase, cytochrome C oxidase, and others. Other active compounds resulting from fermentation are iron-containing proteins and other ferrous compounds.

The fermentation products result from fermentation, mainly of carbohydrates, like glucose, fructose, galactose, mannose, xylose, and their homo- and hetero-oligomers, such as sucrose, maltose and especially lactose, but also of proteins. Such carbohydrates and proteins may be present in common food products such as milk, fruits, corn, vegetables. The fermented food products, such as buttermilk, yoghurt, acido- philus milk, beer, wine, honey, sauerkraut, fermented whey etc. may also be used as such. The amount of products released by microbial fermentation may be from 50 mg to 5 g of dry matter derived from microbial fermentation per daily dosage. In case of fermented whey and similar fermented products, the total daily amount of whey solids and dry fermentation products is preferably 50 mg- 50 g, in particular 100 mg- 10 g. Where the fermentation products are derived from non-whey products such as honey, white wine etc, their level can be related to the dry matter of these non-whey products, which are used then at levels of e.g. 50 mg to 20 g.

The effectiveness of the protective combinations can be determined using a monitoring compound. The system developed allows the measurement of the actual activity of scavengers in vitro. In this system antipyrine or another aniline derivative is used as a probe, because it reacts sufficiently fast with radicals and gives unique detectable products upon reaction with a radical. Radicals are formed by gamma- irradiation of a solution with antipyrine. The aniline compound has the general formula R^N- r , wherein R¹ and R² are each C,-C₆ alkyl, C,-C₆ alkenyl, C,-C₆ acyl or C,-C₆ alkylamino, or together with the nitrogen atom form a 5- or 6-membered ring which may contain one or more further nitrogen, oxygen or sulphur atoms. Preferably R¹ represents an acyl group and more preferably R*R²N- represents an optionally substituted oxopyrrolyl, oxoimid- azolyl, oxopyrazolyl, oxo(is)oxazolyl, oxo(iso)thiazolyl, oxopyridyl, oxopyridazinyl or oxo-oxazinyl ring or di- or tetra-hydro analogue thereof. Most preferably, the compound is antipyrine (R*R²N- is a l,5-dimethyl-3-oxo-2-pyrrazolinyl group).

Either the disappearance of antipyrine can be followed or the concentration of formed molecules/radicals can be measured by known techniques as chromatographic methods, for example micellar electrokinetic chromatography, HPLC, GLC, capillary chromatography, autoanalyser methods and direct radical measurements like electron spin resonance. The nature of the o- and m-hydroxy derivatives can be established, if necessary, by spectrometric methods, such as NMR. Ligand assays or colour tests can be used as well.

To a solution of antipyrine a solution containing a potential scavenger is added, which is subsequently irradiated. If one measures the concentration of antipyrine, or the concentration of the degradation products, upon gamma-irradiation, it is found that the relative rate of degradation of antipyrine is slowed down by the scavenging power of the potential scavenger solution.

Typically 10^"3M of antipyrine is used and for irradiation a °°Co gamma source. To the solution an equal amount of a 10^"3M solution of scavenger is added and the mixture is irradiated with in total 4 kGy, at a rate of 1.5 kGy h. The conversion of antipyrine is measured and compared with the same mixture without scavenger or with water instead of a scavenger, in case a solvent of unknown composition is tested. Other methods of forming radicals can be used and other compounds than antipyrine, for example other aromatic xenobiotics with unsubstituted ortho- and meta-positions such as phenobarbital but also salicylic acid. The advantage of using antipyrine however is that it is not present in food or the body (for in vivo and in vitro applications) and therefore no undefined basic level has to be subtracted as is the case for e.g. salicylic acid. Furthermore, antipyrine passes all organs with an optimal concentration ratio to blood. The chosen concentrations are chosen for convenience of detection but can easily be adjusted to other values. Further experimental details are described by Coolen et al in /. Chromatography A, 788 (1997) 95-103.

The invention relates in particular to such a method for determining the risk of radical damage in a human or animal body, to be performed in conjunction with administering a physiologically acceptable aniline derivative, in particular antipyrine, to said body and taking a fluid sample from said body, said method comprising monitoring the level of said aniline derivative and its o-hydroxy and m-hydroxy substituted analogues in said fluid sample. The aniline derivative is a derivative as described above, especially antipyrine. The body fluid may be blood, serum, but especially urine.

For comparison, several known scavengers were measured such as vitamin E and vitamin C. Thereafter a range of food products was tested with different outcomes. Some examples, which had a good radical scavenging potency when compared to vitamin C, are white wine (an Elzasser white wine was tested), red wine, onion extracts and also whey and whey proteins. All these products are decent scavengers in the order of vitamin C, but not more potent. Some comparative examples are given in Table 1. Water is used as a reference and therefore set equal to 100%.

Table 1

Food -product Antipyrine conversion (%,

Water 100 (per definition)

Caseins (2 wt.% solution) 41

Whey proteins (2 wt.% solution) 38

Milk 16

Raw whey 10

Buttermilk 0

Fermented whey 0

Whey and white wine 0

Whey and honey 0

The scavenging effect of whey, when compared to milk or a solution of whey proteins, is surprisingly strong. The effect of buttermilk, fermented whey or whey in combination with white wine (Elzas) is even stronger, in fact 100%. The above strongly suggests that the scavenger in whey does not occur in the raw milk, but has been formed during the (lactic acid) fermentation. In general it was found that fermented milk products had a higher scavenging potency than the milk they were produced from. This may be due to special metabolites or active proteins like enzymes formed from the (lactic acid) fermentation. The synergetic effect of whey and white wine cannot be explained by simple addition of effects and the same holds for the extremely effective protection of antipyrine by the whey combinations and buttermilk. These findings form the essential part of the invention.

The fact that besides white wine also honey was 100% effective indicates that also other combinations with whey can be very effective. Other combinations of whey solids with fermentation products have similar scavenging activity as the combination white wine/whey and whey/honey.

The mixtures of white wine or honey and whey or other fermented (milk) products or fermented vegetable products and/or products with a probiotic action based on microbial activity or the products in itself are acceptable as food products. It might however be necessary to purify, at least partially, the sources in case one would like to use the combined purified or concentrated fractions of scavengers in health applications such as clinical food, medical food and/or functional food.

Clinical foods, containing whey or other fermented (milk) products and white wine or honey or their combined active fractions or similar combinations and their fractions, can be used in case of e.g. radiotherapy, chemotherapy, combination therapy for AIDS, intensive care and post-operative care. Blood cells are protected during combination therapy for AIDS by (fermented) whey, as observed ex vivo.

Medical foods, containing whey or other fermented (milk) products and white wine or honey or their combined active fractions or similar combinations or their fractions, can be used in case of e.g. diabetic patients, patients with a "leaky" intestine, allergy patients, patients with a neurodegenerative disease, cancer patients and patients with cardiovascular problems.

Functional foods, containing whey or other fermented (milk) products and white wine or honey or their combined active fractions or similar combinations or their fractions, can be used in case of e.g. stress situations, sport and exercise and by elderly people. These foods are also part of the present invention.

Another application, which can also be used in addition to in vitro screening for scavengers, is the use of antipyrine or similar compounds, as mentioned above in in- vivo diagnostics allowing the measurement of radical levels and changes therein by detecting the radicals formed from antipyrine upon reaction with radicals. In this case patients or other people, like sportsmen for whom it might be useful to measure the radical levels in the body, are given antipyrine.

The oxidative radicals in the body will react with antipyrine forming o-hydroxy and m-hydroxy phenolic products, which are different from enzymatic metabolites such as p-hydroxyphenol, all of those compounds can be detected after being expelled from the body. The results of such studies can be used to measure the in vivo effect of food products which are supposed to diminish the radical levels, enabling quantitative intervention studies, or medicines having a potentially positive or negative effect

(negative side-effect) on the radical levels in the body.

Having an indication of the radical levels and their variations in a particular person would allow for the design of a diet for people in order to diminish the radical concentrations as a consequence of illness or activities for a particular person or a group of people. Repeated testing yields information whether the tailor-made diet has been effective for an individual patient, in case of clinical and medical foods, as well as for functional foods.

Claims

1. Combined use of whey solids and products released through fermentation by microorganisms, for preparing a foodstuff or medicament for the protection of the human or animal body against the effect of radicals.

2. Use according to claim 1, wherein the whey solids comprise whey proteins.

3. Use according to claim 1 or 2, wherein the microorganisms are selected from lactic bacteria and yeasts.

4. Use according to any one of claims 1-3, wherein the products released by microbial fermentation comprise oxidoreductases and/or iron-containing compounds.

5. Use according to any one of claims 1-4, wherein a daily dosage of 200 mg to 60 g, in particular 1-30 g of whey solids is used.

6. Use according to any one of claims 1-4, wherein a daily dosage of 50 mg to 50 g, in particular 100 mg-10 g of fermented whey solids is used.

7. Method for determining the risk of radical damage in a human or animal body, to be performed in conjunction with administering a physiologically acceptable aniline derivative to said body and taking a fluid sample from said body, said method comprising monitoring the level of said aniline derivative and its o-hydroxy and m- hydroxy substituted analogues in said fluid sample.

8. Method according to claim 7, wherein said aniline derivative is antipyrine.