WO2021025557A1

WO2021025557A1 - Mutant tomatoes and use thereof for preventing or treating vitamin b12 deficiency.

Info

Publication number: WO2021025557A1
Application number: PCT/NL2020/050499
Authority: WO
Inventors: Arie-Dirk Van Der Windt
Original assignee: Hw Innovations B.V.
Priority date: 2019-08-06
Filing date: 2020-08-06
Publication date: 2021-02-11
Also published as: NL2023609B1

Abstract

The invention relates to the field of nutrition and health related products. In particular, it relates to means and methods for preventing and/or treating vitamin B12 deficiency and disorders related thereto. Provided is a tomato plant, fruit, fragment or extract of a ripening-impaired mutant tomato carrying the rin gene, for use in a method of increasing the serum vitamin B12 level in a mammal, for example in a method for preventing and/or treating vitamin B12 deficiency in a mammal.

Description

Title: Mutant tomatoes and use thereof for preventing or treating vitamin B12 deficiency.

The invention relates to the field of nutrition and health related products. In particular, it relates to means and methods for preventing and/or treating vitamin B12 deficiency and disorders related thereto.

Vitamin B12, also known as cobalamin, is a water-soluble vitamin that is involved in the metabolism of every cell of the human body: it is a cofactor in DNA synthesis, and in both fatty acid and amino acid metabolism. It is particularly important in the normal functioning of the nervous system via its role in the synthesis of myelin, and in the maturation of developing red blood cells in the bone marrow.

Vitamin B12 is one of eight B vitamins; it is the largest and most structurally complex vitamin. It consists of a class of chemically related compounds (vitamers), all of which show physiological activity. It contains the biochemically rare element cobalt (chemical symbol Co) positioned in the center of a corrin ring. The only organisms to produce vitamin B12 are certain bacteria, and archaea. Some of these bacteria are found in the soil around the grasses that ruminants eat; they are taken into the animal, proliferate, form part of their gut flora, and continue to produce vitamin B12.

Vitamin B12 is naturally found in animal foods, including meats, fish, poultry, eggs and dairy. The richest sources are liver, clams, kidneys and oysters. As it generally held to be absent from the plant foods (except for sea vegetables like edible algaea) and obtained mostly from animal protein, it is more difficult for vegetarians and vegans to obtain vitamin B12 from their normal diet. It is found in some fermented foods, such as tempeh and more commonly in nori and nutritional yeast (or yeast extract like Marmite). Because there are no common vegetable sources of the vitamin, vegans must use a supplement or fortified foods for B12 intake or risk serious health consequences. Examples of are vegan and vegetarian foods that are fortified with vitamin B12 include some plant milks and breakfast cereals.

Normal serum B12 levels are 145-570 pmol/L, with levels of less than 130 pmol/L typically being said to indicate clinically significant deficiency. However, serum B12 levels are a relatively insensitive determinant of B12 deficiency in that only 50% of patients with clinically confirmed B12 deficiency have levels less than 130 pmol/L, 40% are 130-250 pmol/L, and at least 5-10% have values in the 250-350 pmol/L range.

For people over 14, the recommended daily intake (RDI) for vitamin B12 is 2.4 meg, and most people meet this requirement through diet. Pregnant women have slightly higher vitamin B12 needs than the general population. Low maternal levels of this vitamin have been associated with birth defects in infants. Additionally, a large systematic review showed that B12 deficiency is associated with a higher risk of premature birth and low birthweight in newborns. Therefore, the RDI for vitamin B12 during pregnancy is 2.6 meg. Vitamin B12 deficiency in breastfed infants has been linked to developmental delay and B12 deficiency in infants can lead to irritability, decreased appetite and failure to thrive. For these reasons, the RDI for this vitamin for breastfeeding women is higher than that for pregnant women — namely 2.8 meg.

Vitamin B12 recommendations do not differ for people following a plant-based diet. However, the RDI of 2.4 meg for people under 50 years of age is much harder to meet on a vegetarian or vegan diet. In a review of 40 studies on vitamin B12 in vegetarians, up to 86.5% of vegetarian adults — including older adults — were found to have low levels of vitamin B12. The most significant difference between B12 and other B group vitamins is that B12 can be stored in the body, specifically in the muscles and liver. For this reason deficiency usually develops after 5 to 10 years of not consuming dietary B12. Unfortunately, B12 deficiency is common, especially in the elderly. People at risk of a B12 deficiency include the elderly, subjects that had surgery that removes the part of the bowel that absorbs B12, subjects on the drug metformin for diabetes, people following a strict vegan or vegetarian diet, and those taking long-term antacid drugs for heartburn.

A serious vitamin B12 deficiency can be corrected by prescription medication. Vitamin B12 supplements are available in single agent or multivitamin tablets; and pharmaceutical preparations may be given by intramuscular injection. Vitamin B12, in the form of cyanocobalamin and occasionally hydroxocobalamin, can be administered parenterally as a prescription medication, usually by (weekly) intramuscular injection. Parenteral administration is typically used to treat vitamin B12 deficiency caused by pernicious anemia and other conditions that result in vitamin B12 malabsorption and severe vitamin B12 deficiency. Vitamin B12 is also available as a prescription medication in a gel formulation applied intranasally, a product marketed as an alternative to vitamin B12 injections that some patients might prefer. This formulation appears to be effective in raising vitamin B12 blood levels, although it has not been thoroughly studied in clinical settings. A mild B12 deficiency can be corrected with a standard multivitamin supplement.

However, the administration of injections is painful and cumbersome, and the intake of pills, oral supplements and fortified food items is gradually loosing acceptation and appreciation by the consumers, in particular those wishing to follow a more natural diet. It would therefore be desirable if vitamin B12 intake or supplementation can be achieved by a plant-based food product which is ordinarily ingested by a mammal, in particular a human being. Preferably, the vitamin B12 providing food product furthermore satisfies one or more of the following criteria: (i) it has a palatable taste; (ii) is of low caloric value; (iii) of non-animal origin; (iv) non-transgenic, (v) easy to obtain and (vi) economically attractive.

The present inventors surprisingly observed that ripening-impaired mutant tomatoes of the rin type contain significant amounts of vitamin B12, and that the rin content of a tomato correlates positively with its vitamin B12 content. Furthermore, the vitamin B12 content was found to increase upon exposure of rin tomato fruits to light. Notably, tomatoes carrying the nor gene, a different type of ripening impaired mutant tomato, did not show a detectable vitamin B12 level at any stage of development. Oral ingestion of an acceptable serving size of rin tomato fruits was found to significantly increase the serum vitamin B12 level in a human subject.

Accordingly, in one embodiment the invention provides a tomato plant, fruit, fragment or extract thereof for use in a method for preventing and/or treating vitamin B12 deficiency in a mammal, wherein the tomato is a ripening-impaired mutant that carries the ripening inhibitor (rin). A further embodiment relates to a tomato plant, fruit, fragment or extract of a ripening-impaired mutant tomato carrying the rin gene, for use in a method of increasing the serum vitamin B12 level in a mammal. Also provided is a ripening inhibitor (rin) mutant tomato plant, fruit, fragment or extract thereof for use as source of vitamin B12. As used herein, the term ‘’vitamin B12” is used to describe compounds of the cobalt corrinoid family, in particular those of the cobalamin group.

The most used compound of this group is cyanocobalamin and as such the term vitamin B12 is sometimes used to refer to cyanocobalamin. In this specification the term vitamin B12 should be attributed its broad meaning so as to include all the cobalt corrinoids of the cobalamin group, which include in particular cyanocobalamin, hydroxocobalamin, methylcobalamin and characterized by a cyano, hydroxyl, methyl or radical respectively. The methylcobalamin and 5'desoxyadenosylcobalamin compounds are known to be unstable to light in isolated form and are easily transformed to hydroxocobalamin in aqueous solution.

The tomato ( Lycopersicon esculentum) is a plant from the Solanaceae family (Solanaceae) native to America and cultivated all over the world for its edible fruit. Said fruit is a very coloured berry, with shades that typically change from yellowish to red, due to the presence of the lycopene and carotene pigments. It has a lightly acidic flavour, a diameter of 1 to 2 cm in the wild species, and is much bigger in the cultivated varieties. It is produced and consumed all over the world both fresh and processed in different ways, e.g. as a sauce, puree, juice, dehydrated or canned. The tomato is a food having a low quantity of calories. In fact, 100 grams of tomato only have 18 kcal. Most of its weight is water and carbohydrates are the second important constituent. It contains simple sugars that confer it a light sweet flavour and some organic acids that provide the characteristic acidic flavour. The tomato is an important source of certain minerals as potassium and magnesium. From its vitamin content, the vitamins Bl, B2, B5 and C are often highlighted. Notably however, the presence of vitamin B12 in any (mutant) tomato has heretofore never been taught or suggested in the art. Hence, the present finding that a rin tomato contains significant amounts of vitamin B12 that can be absorbed by the human body is highly unexpected.

The ripening inhibitor gene (rin) is a semi-dominant tomato gene which was first described in 1968 by Robinson and Tomes, "Ripening Inhibitor: A Gene with Multiple Effects on Ripening." Rpt. Tomato Genetics Cooperative 18:36-37. The rin gene is available from several sources, including the C. M. Rick Tomato Genetic Resource Center (TGRC) at the University of California, Davis. The rin gene is described in the literature, e.g., in Davies et al. mentioned above.

Tomato plants harboring the rin mutation yield fruits that fail to ripen. Tissue softening and pigment synthesis which occur in normal tomato fruits are inhibited in fruits of rin tomato mutants (Davies et al. 1981, “The Constituents of Tomato Fruit — The Influence of Environment, Nutrition and Gene Type,” CRC Critical Reviews in Food Science and Nutrition, 15:205- 280), indicates that the deleterious effects of ripening inhibitor genes in the heterozygous state may possibly be overcome by incorporating genes which will enhance color, such as high pigment and crimson.

Additionally, rin plants display enlarged sepals and loss of inflorescence determinacy. Positional cloning of the rin locus revealed two tandem MADS-box genes (LeMADS-RIN and LeMADS-MC), whose expression patterns suggested roles in fruit ripening and sepal development, respectively. The rin mutation alters expression of both genes. Gene repression and mutant complementation demonstrate that LeMADS-RIN regulates ripening, whereas LeMADS-MC affects sepal development and inflorescence determinacy. LeMADS-RIN demonstrates an agriculturally important function of plant MADS-box genes and provides molecular insight into nonhormonal (developmental) regulation of ripening (Vrebalov et al. Science 12 April 2002: Vol. 296 no. 5566 pp. 343-346). US 4,843,186 relates to a heterozygous tomato plant resulting from crossing a male parent containing the Rin/Rin gene with a female parent, said tomato plant having a rin gene from the male parent as a heterozygote and having the characteristics of a very firm fruit with excellent tomato-like taste having a shelf like of at least two weeks, without substantially deteriorating taste or firmness, and developing a full red color.

US 2003134026 discloses homozygous rin and/or nor tomatoes, or tomatoes heterozygous in both rin and nor are used to prepare a tomato paste, juice or sauce having good viscosity as well as good color.

EP2882280B1 / WO2014/027886 relates to the use of ripening- impaired mutant tomatoes, such as a ripening inhibitor (rin), nonripening (nor), and /or never ripe (Nr) tomatoes for inhibition, amelioration or prevention of adipogenesis mediated diseases such as obesity, lipid storage disease and hyperlipemia.

Thus, the prior art fails to disclose or suggest the presence of Vitamin B12 in a ripening-impaired rin mutant tomato, extracts or purified fractions thereof. More specifically, its beneficial application in the prevention or suppression of disorders relating to vitamin B12 deficiency was heretofore never recognized.

The mutant tomato for use in the present invention carries the rin gene. As used herein, a ripening inhibitor (rin) mutant tomato refers to a Lycopersicon esculentum expressing the mutant ripening inhibitor (rin) tomato gene, thus encompassing any variety in the spectrum ranging from heterozygous rin to homozygous rin tomatoes. In one embodiment, the tomato is heterozygous for rin. In another embodiment, the tomato is a homozygous rin tomato. Using tissue culture techniques, the present inventors succeeded in generating tomato varieties having a rin content from between 50 to 100%. As used herein, the term "rin content” is defined as the amount of the tomato rin mRNA relative to the arbitrary amount of 100% rin in a rin/rin homozygous tomato. The rin content of a rin heterozygote is 50%. The rin content of a RIN homozygote is 100%.

In one embodiment, the tomato has a rin content of at least 50%, preferably at least 65%, more preferably 70-100%. In another embodiment, the tomato is homozygous for rin. In contrast to other ripening impaired tomatoes, such as those carrying the nor or Nr gene, a mutant rin tomato can develop into fruits that are acceptable (e.g. in taste and appearance) for human consumption.

The rin content is suitably determined by methods known in the art based on the known sequence of the rin gene. For example, standard PCR methodology can be used based on a set of primers designed to specifically detect the rin mutation. Quantitative real-time PCR (RT-PCR) is preferred. RT-PCR is currently the standard method for accurate expression profiling of a moderate number of selected genes, its main advantages being a high sensitivity and specificity, and a broad quantification range. RT-PCR protocols are well known in the art. See for example Derveaux et al. Methods 50 (2010), 227-230. Regardless of the experimental technique employed, appropriate normalization is essential for obtaining an accurate and reliable quantification of gene expression levels. The purpose of normalization is to correct for variability associated with the various steps of the experimental procedure, such as differences in initial sample amount, RNA recovery, RNA integrity, efficiency of cDNA synthesis, and differences in the overall transcriptional activity of the tissues or cells analyzed. Preferably, the PCR analysis also involves detection of one or more tomato housekeeping genes or constitutionally expressed genes such as GAPDH, EFal, TBP, RPL8, APT, DNAJ, TUA, TIP41, SAND, CAC and SGN- U346908SGN (Exposito-Rodriquez et al. (BMC Plant Biology 2008, 8:131). In addition to detecting the rin gene, the relative expression level of one or more rin target genes may be determined. For example, the rin/rin mutant was found to lack expression of LeACS2, LeACS4, TBG4, LeEXPl, LeMAN4 and PSY1, and shows decreased expression levels of LeACOl, ETR3, PG and INV, while these genes are highly upregulated in the wild-type fruit, indicating that all of these genes are regulated directly or indirectly by rin (Fujisawa et al., BMC Plant Biology 2011, 11:26).

In one embodiment, the tomato has a rin content of at least 53%, like at least 60%, preferably at least 65%, more preferably at least 70%. For example, tomatoes may have a rin content of 75-100%.

In one embodiment,, the tomato is selected from the group consisting of the varieties ‘’Carrasco’ and "336.485” {rin content 50%), and ‘Tomango” {rin content 100%). In a specific aspect, the rin mutant tomato, plant, fruit, fragment or extract thereof is a Carrasco tomato plant, fruit, fragment or extract thereof.

As indicated above, the invention can be practised using a rin mutant tomato, plant, fruit, fragment or extract thereof. As used herein, "fragment” refers to a subsection of the plant, like a leave, stem, root, or of the fruit. As is shown herein below, high amounts of vitamin B12 were found in fruits, leaves and roots of a rin tomato. In one embodiment, the root, leave or fruit is used. Preferably, the fruit or a fruit extract is used. For the ease of consumption and customer acceptance and appreciation, the use of whole tomato fruits is particularly preferred. For example, it is envisaged that the rin tomato is consumed as part of the normal human diet, e.g. as fresh fruits or a processed product thereof like a paste or sauce, thus replacing at least part of the traditional tomato with the benefit of supplementing vitamin B12.

The mutant tomato can vary in size, from tomberries, about 5 mm in diameter, through cherry tomatoes, about the same 1—2 centimeters (0.4— 0.8 inches) size as the wild tomato, up to beefsteak tomatoes 10 centimeters (4 in) or more in diameter. The most widely grown commercial tomatoes tend to be in the 5—6 centimeters (2.0— 2.4 in) diameter range. Tomatoes grown for canning and sauces are often elongated, 7—9 centimeters (3—4 in) long and 4—5 centimeters (1.6— 2.0 in) diameter; they are known as plum tomatoes, and have a lower water content.

In one embodiment, the invention provides the use of a rin mutant tomato extract, paste, sauce or juice, to increase or maintain vitamin B12 levels in a mammal, in particular to increase or maintain serum vitamin B12 levels. The tomato extract can be obtained by, for example, extracting raw material tomatoes with a suitable solvent. In another embodiment, the tomato fruit is used as a paste or in a dried form.

The raw rin material tomato to be extracted may be the whole tomato plant, as well as the fruit, pericarp, juice or any other arbitrary parts. In case that the raw material tomato contains a large amount of moisture, it is preferably used after drying in order to improve efficiency. More specifically, the moisture content may be reduced by, for example, drying either naturally or with hot air for 1 to 24 hours at 50 to 150°C. Good results are obtained when raw material is freeze-dried prior to extraction. In addition, in order to increase extraction efficiency, the raw material tomato is preferably used after being finely ground. There are no particular restrictions on the grinding means, and examples of such include a method using a mortar, and methods using a crushing machine such as a whirling blender or a homogenizer. The raw material tomato is preferably ground to a size of 16 mesh or finer.

The invention also provides the use of a ripening-impaired rin tomato, plant, fruit, fragment or extract thereof for supplementing or maintaining the vitamin B12 level in a mammal. In one embodiment, the invention provides a rin tomato fruit or extract thereof for use in a method for preventing or treating a disease conditions associated with vitamin B12 deficiency in a mammal. Preferably, the mammal is human or an animal. Thus, veterinary applications of the mutant tomato are also envisaged.

Preferably however, the mammal is a human individual, in particular a human individual that is at increased risk of being or becoming deficient in vitamin B12. Whole fruits of a rin tomato are easily incorporated in the human diet. People at risk of a B12 deficiency include the elderly, subjects that had surgery that removes the part of the bowel that absorbs B12, subjects on the drug metformin for diabetes, pregnant women, breastfeeding women, people following a strict vegan or vegetarian diet, and those taking long-term antacid drugs for heartburn.

In a specific embodiment, the invention provides a rin tomato fruit or extract thereof for use in a method for preventing or treating a disease conditions associated with vitamin B12, such as Biermer anemia and the para-Biermer anemias which are cobalamino-curable, further neuritis and polyneuritis and disorders of the protein metabolism.

The vitamin B12 status in human subjects is typically assessed via serum or plasma vitamin B12 levels. Values below approximately 170—250 pg/mL (120—180 picomol/L) for adults indicate a vitamin B12 deficiency. Accordingly, in one embodiment the human subject to be treated with rin mutant tomato has a vitamin B12 value below approximately 170—250 pg/mL (120—180 picomol/L). However, evidence suggests that serum vitamin B12 concentrations might not accurately reflect intracellular concentrations. Elevated methylmalonic acid levels (values >0.4 micromol/L) might be a more reliable indicator of vitamin B12 status because they indicate a metabolic change that is highly specific to vitamin B12 deficiency.

Also provided is a method for treating vitamin B12 deficiency in a subject comprising administering to the subject a (therapeutically) effective amount of a ripening-impaired rin tomato plant, fruit, fragment or extract thereof, preferably a fresh rin tomato fruits. The subject is preferably a human subject. The formulation is preferably administered to the subject 1-4 times daily. The formulation is typically administered for at least 2 weeks, preferable at least one month.

The amount of a rin tomato plant, fruit, fragment or extract thereof to be administered can vary according to specific needs or desired effects. It was found that the daily intake of about 3-4 medium sized tomatoes (i.e. about 70-100 grams) of a rin heterozygote was sufficient to significantly increase the serum vitamin B12 level in a human being of approximately 70 kgs. For example, the serum level was increased with about 20% after a daily intake of 100 gram rin tomato fruits during only a one week trial period. Hence, in one embodiment the equivalent of at least 0.5 gram, preferably at least 0.75, more preferably at least 1 gram of rin tomato fruit per kg bodyweight is used for daily dosage or consumption. Of course, the equivalent amount can be adapted accordingly where a less or a more frequent dosage is intended.

EXPERIMENTAL SECTION

Example 1: Determination of rin content

Using an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), total RNA was extracted and purified from tomato fruits of varieties having different rin contents, including a normal (a genotype of RIN/RIN) plant, (mature green, pink coloring and red ripe) and of a rin mutant (rin/rin) plant at periods corresponding to these stages, as previously described (Kitagawa et ah, Characterization of tomato fruit ripening and analysis of gen expression in FI — Hybrids of the ripening inhibitor ( rin ) Mutant. Physiol Plantarium 2005 ,123 (3) :331-338). Complementary DNA was synthesized from total RNA using a PrimeScript II first cDNA strand synthesis kit (Takara Biotech) and then applied in real-time PCR as a template. Expression levels of the rin genes were analyzed by quantitative real-time reverse transcription PCR (qRT-PCR) using oligonucleotide primers specific for rin (GenBank accession number AF448522).

Example 2: Determination vitamin B12 content using a microbiological assay.

Tomato fruits were into tomato juice (puree) with a blender / ultraturrax after careful washing with water and drying. 1.0 g of this juice was mixed with 20 ml of 0.1 M acetate buffer (pH = 4.5) and 0.25 ml of 1% potassium cyanide solution. After heating to 95 ° C in a water bath (30 minutes) and cooling, it was brought to a 40.0 ml with water.

For use in the microbiological assay, the extract was sterile filtered by 0.22 pm (hydrophilic Teflon filter). The microbiological determination was then carried out with a commercially available VitaFast B12 test kit (R- Biopharm GmbH).

In this test kit, either blank, standards (positive control), extract or B12 standard spiked extract are added to a culture medium containing the Lactobacillus delbrueckii subsp. lactis. This bacterium needs vitamin B12 for its growth. Because the culture medium itself does not contain vitamin B12, any bacterial growth therefore depends on the B12 from the added sample (or standards). After incubation for 48 hours (37°C), the turbidity of the culture medium at 610 nm is measured spectrophotometrically as a measure of growth.

A clear growth was observed in both the tomato sample and the spiked tomato sample (internal control for possible growth-inhibiting disturbances). Positive (standards) and negative (blank) controls were in accordance with the manufacturer's test kit manual to provide a standard curve in the range of 0.03 - 0.18 microgram vit B12/100 ml. For samples from Carrasco tomato fruits (50% rin), values in or just above the upper limit of the standard curve were obtained.

It is important to mention that only a heating step has been used in the processing of the tomato extract. No other reagents have been added, such as, for example, diastase enzymes. These may themselves still contain traces vitamin B12 from their production media, and may thus cause a false positive result. Due to this chosen method, the level of vitamin B12 found (or that of highly related corrines with B12 activity) can only originate from the tomatoes.

Example 3: Determination vitamin B12 content using an immunological assay.

This example describes the determination of vitamin B12 levels using an immunological approach. Prior to immunological analysis, the sample must first be purified and concentrated. To this end, 100 g of tomato fruits was processed after careful washing with water and drying with a blender / ultraturrax to form a tomato juice (puree). To this juice, 50 ml of 1 M acetate buffer (pH 4.8) and 2 ml of 0.5% potassium cyanide solution were added. After heating at 95°C in a water bath (60 minutes) and cooling, it was filtered through a paper filter (S&S 595).

Purification was performed using a column comprising Amberlite™ XAD-4 resin, with a bed size of 8 cm². The resin was first pre-rinsed with 0.1 M potassium hydroxide in methanol until the extinction at 254 nm was less than 0.1. After equilibration with water, the entire tomato filtrate was passed dropwise over the column. Then, the column was washed with 160 ml of water, and after careful blow-drying, eluted dropwise with 80% methanol. The resulting eluate was concentrated in vacuo, and taken up in 5.0 ml of methanol.

The subsequent immunological determination was performed using a clinical B12 analyzer (AD VIA - Siemens). To this end, 100 mΐ of the methanol solution was subjected to solvent evaporation under argon, and the resulting residue was taken up in 1 ml of human serum with a known vitamin B12 concentration. The difference between the measured value and the known value of the serum thus gives the concentration in the tomato sample. This approach was chosen because during initial experiments it was found that tomato samples diluted in physiological salt lead to disturbances in the assay.

The clinical assay method employs a recombinant vitamin B12 binding protein, which is identical to the vitamin B12 binding transport protein from the stomach. After reaction with the vitamin B12 present in the sample (if any), the concentration of this entire complex is determined. In this manner, the assay only detects the concentration of purely functional B12, i.e. the vitamin B12 which can actually be absorbed by binding to the transport protein. Corrins related to vitamin B12, which cannot be absorbed via the transport protein, are therefore not co- determined in this approach.

The results of this assay revealed that the Aim-mutant tomato Carrasco has a vitamin B12 content in the range of 0.05-0.09 microg/15 g tomato fruits. In contrast, no detectable vit B12 was found in wildtype (0% Rin) or a Nor- mutant tomato (data not shown). Example 4: Cobalt measurements in the purified and concentrated extract

Because the central cation of the vitamin B12 molecule is a cobalt atom, the cobalt content of the purified and concentrated eluate were also investigated as a further indicator of vitamin B12.

To this end, 100 mΐ of the methanol solution after evaporation of the solvent under argon of Example 3 was taken up in 2 ml of measurement buffer to obtain a test solution. An aliquot of the test solution was subjected to ICP- MS in order to determine the cobalt content.

As expected, the tomato eluate was found to give a greatly increased cobalt value compared to a background control sample (methanol).

Strictly speaking, the ICP-MS assay cannot distinguish between free cobalt (as mineral from the tomato itself) and cobalt bound in the vitamin B12 molecule. However, the binding to XAD-4 resin is mainly based on hydrophobic interaction and much less based on ion exchange. For this reason, the cobalt in the eluate is more likely to come from binding to a large hydrophobic molecule (such as the B12 corrin skeleton) than from a direct interaction of free cobalt with the XAD-4 resin.

Example 5: In vivo vitamin B12 supplementation

This example demonstrates that vitamin B12 present in mutant rin tomato is effectively absorbed by the human body upon oral ingestion.

A healthy human volunteer (male; age 59) consumed a daily amount of 100 gram of ripened fruits of the rin heterozygote tomato variety ‘’Carrasco” for seven consecutive days. Care was taken that the remaining diet did not contain large amounts of meat, eggs, or other products known to be enriched in vitamin B12.

A blood sample was taken prior to, and on day 7 of the trial period. Analysis of the vitamin B12 content in the serum using standard clinical methods revealed that the basal level of 426 pmol/L was increased to 506 pmol/L i.e. an 18% increase, which is comparable to the effect of the conventional weekly intake of prescription vitamin B12 tablets.

Claims

1. A tomato plant, fruit, fragment or extract of a ripening-impaired mutant tomato carrying the rin gene, for use in a method of increasing the serum vitamin B12 level in a mammal.

2. A tomato plant, fruit, fragment or extract of a ripening-impaired mutant tomato carrying the rin gene, for use in a method of preventing and/or treating vitamin B12 deficiency in a mammal.

3. Tomato plant, fruit, fragment or extract thereof for use according to claim 1 or 2, wherein the mutant tomato has a rin content of at least 50%.

4. Tomato plant, fruit, fragment or extract thereof for use according to any one of claims 1-3, wherein the tomato is selected from the group consisting of the varieties ‘Carrasco”, ‘’336.485” and ‘ omango”.

5. Tomato plant, fruit, fragment or extract thereof for use according to any one of the preceding claims, wherein the fruit or fruit extract is used.

6. Tomato fruit extract according to claim 5, wherein the extract is prepared by extraction a tricarboxylic acid, preferably an acetate buffer in the range pH 4-5.

7. Tomato plant, fruit, fragment or extract thereof for use according to any one of the preceding claims, wherein the mammal is at increased risk of developing a disease condition associated with vitamin B12 deficiency.

8. Tomato plant, fruit, fragment or extract thereof for use according to any one of the preceding claims, wherein the mammal is a human individual.

9. Tomato plant, fruit, fragment or extract thereof for use according to claim 8, wherein the human individual follows a vegan or vegetarian diet.

10. A method for increasing the blood serum level of vitamin B12 in a subject, comprising administering to the subject an effective amount of a ripening inhibitor ( rin ) mutant tomato plant, fruit, fragment or extract thereof.

11. Method according to claim 10, comprising administering a daily amount of at least 30 grams, preferably at least 50 grams of a ripening inhibitor (rin) mutant tomato fruit.

12. A ripening inhibitor (rin) mutant tomato plant, fruit, fragment or extract thereof for use as source of vitamin B12.