IE20160221A1 - Process for modifying and/or increasing Vitamin D content of mushrooms - Google Patents

Abstract

The present invention relates to a process for modifying and/or increasing the Vitamin D content of mushrooms comprising a process for subjecting pre-selected mushrooms to UV irradiation to result in a defined Vitamin D content.

Description

Background to the Invention Vitamin D is an essential fat soluble vitamin known to contribute to normal absorption and utilisation of calcium and phosphorous, and to contribute to the maintenance of normal bones, teeth and normal muscle function. Vitamin D is also involved in the normal function of the immune system and contributes to the process of cell division. Vitamin D is produced within the skin when exposed to sunlight. UVB light stimulates the production of Vitamin D3 from 7-dehydrocholesterol in the skin. Very few foods contain significant amounts of Vitamin D, these include fish and eggs and fortified milk, cereals and juices and wild mushrooms. The RDA in Europe for Vitamin D is 5 pg / day. In the USA (loM, 2010) the RDA is considerably higher (10 to 15 pg / day depending on age).

A diet deficient in Vitamin D in conjunction with inadequate sun exposure causes osteomalacia (or rickets when it occurs in children), which is a softening of the bones. Vitamin D deficiency has become worldwide issue in the elderly and remains common in children and adults. Low blood calcidiol (25-hydroxy-vitamin D) can result from avoiding the sun. Deficiency results in impaired bone mineralization and bone damage which leads to bone-softening diseases. Foods that naturally provide vitamin D include: fatty fish like tuna, mackerel, and salmon; foods fortified with vitamin D like some dairy products, orange juice, soy milk, and cereals; beef liver; cheese: and egg yolks. To supplement these natural sources, the use of Vitamin D supplements or foods enriched with Vitamin D are forming a new market for consumers.

Extensive research has shown that mushrooms exposed to UV-light (continuous or pulsed) can produce Vitamin D. This is assumed to be by converting the ergosterol present naturally in mushrooms into ergocalciferol. The subject of most of these studies is to maximize the increase on Vitamin D of different mushroom species and derived mushroom products and at the same time minimize some other negative effects such as browning on the surface of the mushroom. Levels of Vitamin D claimed are as high as several thousand times the recommended daily intake. However, levels too high of Vitamin D2 can have a detrimental effect for their commercialisation, in particular in jurisdictions where the tolerable levels are set by strict thresholds.

US patent application number 20090269441 shows that pulsed UV-light is preferred in order to enhance vitamin D2 levels while at the same time having a low impact on appearance (i.e. browning). However, the vitamin D levels obtained in this method are too variable and too high to commercialize in Europe and other jurisdictions. In addition there is a high degree of unreliability and variability and the variability is particularly problematic from a quality control point of view. The addition of vitamins and minerals into food must comply with guidelines set in EC Regulation 1925/2006 and EC labelling Regulation 1196/2011. A discussion paper published by the European Commission establishes the maximum safety level (MSL) for Vitamin D in 35 pg a day.

A recently published Scientific Opinion of the EFSA Panel on Dietetic, Nutrition and Allergies (NDA) has reviewed the Tolerable Upper Intake Level of Vitamin D (UL). As shown in Table 1, the UL for vitamin D for adults, including pregnant and lactating women, has been established at 100 pg / day.

Table 1. Summary of Tolerable Upper Intake Levels for Vitamin D Age (years) Tolerable Upper Intake Level (UL) vitamin D(pg / day) Children 0-1 25 1-10 50 11-17 100 Adults > 18 100 Thus, in order to meet the European Food Safety Authority requirements and also to ensure equivalent Vitamin D levels to wild outdoor mushrooms (typically 5 to 35 pg), it is necessary to ensure levels are within the allowable ranges and any process to prepare such enhanced mushrooms provide consistent and repeatable Vitamin D levels.

The present invention is directed towards providing an improved process for increasing the Vitamin D content of mushrooms. Furthermore, the invention aims to provide a refined and consistent method for accurately increasing the Vitamin D content of mushrooms, optionally regardless of their size.

Summary of the Invention According to a first aspect of the invention, there is provided a process for increasing the Vitamin D content of mushrooms to approximately 5 to 40 pg, preferably 5 to 20 pg, Vitamin D per 100 gram, with a precision of approximately 12%, comprising the steps of pre-selecting mushrooms from flush 1, flush 2, or flush 3; measuring the diameter and weight of the mushrooms; calculating surface area to volume ratio, and optionally density, of the mushrooms; selecting mushrooms with a density from approximately 0.2 to 0.7 g/cm3, optionally 0.25 to 0.5 g/cm3: and/or a surface area to volume ratio, or sa/vol, from approximately 0.5 to 2.5 cm'1, optionally 0.6 to 2.5 cm*1; optionally placing a single layer of mushrooms in an open top container wherein the mushrooms are placed with the mushroom cap or pileus facing upwards such that the gill is adjacent to the inside base of the open top container; optionally passing the open top container with the mushroom cap or pileus facing upwards under a UV light source wherein the distance from the inside base of the open top container to the UV light source is from approximately 5 to 30 cm and the mushrooms receive from 1 to 5 pulses of UV irradiation such that the mushrooms receive from approximately 0.6 to 15 m-J/cm2, preferably 0.68 to 15 mJ/cm2, of UV irradiation.

The present invention is also directed to mushrooms with approximately 5 to 40 pg, preferably 5 to 20 pg Vitamin D per 100 gram produced according to the process of the invention.

Detailed Description of the Invention In this specification, it will be understood that any mushroom type may be used. Reference is made to Agaricus bisporus as this is one of the most commonly used commercial mushroom types. Button mushrooms (small), closed cup (medium) mushrooms or Portobello mushrooms (large) may be used in accordance with the invention. It is envisaged that any mushroom, with similar morphological characteristics, such as density and surface to volume ratio, may be used. Pleurotus ostreatus or Lentinula edodes can for example be used in accordance with the invention.

It will be understood that surface area to volume ratio (cm'1) discussed in the specification may be alternately referred to as S/V ratio, SA/V, sa/v and these terms are interchangeable.

According to the first aspect of the invention, there is provided a process for increasing the Vitamin D content of mushrooms to within approximately 5 to 40 pg Vitamin D per 100 gram, with a precision of approximately 12%, comprising the steps of pre-selecting mushrooms from flush 1, flush 2, or flush 3; measuring the diameter and weight of the mushrooms; calculating surface area to volume ratio and density of the mushrooms; selecting mushrooms with a density from approximately 0.2 to 0.7 g/cm3 and a surface area to volume ratio or sa/vol from approximately 0.5 to 2.5 cm'1 optionally 0.6 to 2.5 cm'1; placing a single layer of mushrooms in an open top container wherein the mushrooms are placed with the mushroom cap or pileus facing upwards such that the gill is adjacent to the inside base of the open top container; passing the open top container with mushroom cap or piieus facing upwards under a UV light source wherein the distance from the inside base of the open top container to the UV light source is from approximately 5 to 30 cm and the mushrooms receive from 1 to 5 pulses of UV irradiation such that the mushrooms receive from approximately 0.6-15 mJ/cm2, optionally 0.6 - 5 mJ/cm2,of UV irradiation.

The step of pre-seiecting mushrooms from flush 1, flush 2, or flush 3 (i.e. crop cycle 1, 2 or 3) involves identifying the mushroom from an established size, and removing it from the mushroom bed, for example with a slight twist and trimming the stem, which step is optionally conducted manually by trained pickers.

The step of measuring the diameter involves comparing the mushroom to a standard (pattern) and measuring the weight of the mushrooms is conducted using portable weighting scales.

The step of calculating surface area to volume ratio, and density of the mushrooms can be carried out using the general mathematical formulae for ellipsoids. Density can be calculated by dividing the mass by its volume or alternatively using a pycnometer (measurement of the water volume displacement).

Once the parameters above have been calculated, mushrooms within a specific range are selected, optionally manually selected, and optionally placed inside a punnet and chilled prior to its UVtreatment and final post-harvest packing stage.

The present invention is directed to an improved process to enhance in a controlled manner the nutritional profile of mushrooms, preferably A. bisporus, by increasing the Vitamin D content using a controlled dose of, for example pulsed, UV-light. The dose is controlled using a combination of distance from lamp to mushroom (conveyor belt) and number of pulses. The selected combination depends on the surface to volume ratio of the samples treated. The pulsed UV light increased the vitamin D content with no deleterious effects to mushroom quality nor to its general composition (proximate analysis).

Advantageously, the present invention is directed to a process for producing mushrooms with a consistent level of Vitamin D per 100 gram serving, ideally up to 100 pg Vitamin D per 100 gram serving, preferably up to 50 pg Vitamin D per 100 gram serving, even more preferably from 5-40 pg Vitamin D per 100 gram serving. Thus, the process of the present invention enables in a precise and controlled increase in the levels of Vitamin D in mushrooms, which are complaint with European legislation and equivalent Vitamin D levels to wild outdoor mushrooms (typically 5 to 35 pg). This process can increase Vitamin D levels in smaller quantities in a reliable manner.

The levels of Vitamin D in Agaricus bisporus can be enriched more precisely than those describe previously. This is achieved by taking into account a series of factors included in previous studies such as number of pulses, but also distance from the UV-light source to mushroom container, number of layers, size and position of the mushrooms (gill is not exposed) and positioning of the container under the UV-light source.

Within the ranges defined in the claims, the level of Vitamin D in A. bisporus can be optimized using response surface methodology (RSM), for example, the RSM Statistical program Design Expert version 9.0.3.1 developed by Stat-Ease Incorporated (Minneapolis, USA) may be utilised.

In this method, different mushroom types/sizes are assessed by trained harvesters using a standard to estimate the diameter and a weighting scales to measure the mass. These values are used to calculate surface to volume and density of mushrooms.

The volume bounded by the ellipsoid/mushroom is defined by V, wherein V=(4/3)nabc, wherein a, b, and c are each the length of the semi-principal axes of the ellipsoid.

The surface area of the ellipsoid/mushroom is defined by S, wherein S==4Tr([(ab)1'6+(ac)1'6+(bc)1'6]/3)1/1·6, wherein a, b, and c are each the length of the semi-principal axes of the ellipsoid.

The surface area to volume ratio, and density of the mushrooms is carried out using the general mathematical formulae for ellipsoids. Density can be calculated by dividing the mass by its volume or alternatively using a pycnometer (measurement of the water volume displacement).

Once the mushrooms have been categorised according to these parameters, then are placed inside a punnet into punnets according to different categories of SA/V and density and chilled prior to its UV-treatment and final post-harvest packing stage.

Different mushroom surface to volume ratio (category) leads to a different surface area of the mushroom exposed to the UV-light treatment. Once the surface to volume of the mushroom is selected, then RSM is applied to find out the best combination of distance of the lamp to mushroom contained and the total number of pulses required to attain levels within a specific value.

Higher surface to volume ratio leads to higher vitamin D levels in mushrooms. The surface to volume ratio of mushrooms varies exponentially with the diameter of the mushroom. Baby button mushrooms, closed cup mushrooms and flat mushrooms have well differentiated surface to ratio ranges. Baby button mushrooms have larger surface to volume ratio values and flat mushrooms have a lower surface to volume ratio values. However baby button mushrooms have higher variability in the surface to volume values and flat mushrooms less variability in the surface to volume values. A low variability in surface to volume ratio is important for reducing variability of vitamin D during the treatment.

It is important that other factors such as the number of layers exposed the positioning of the container under the lamp source, the positioning of the mushroom inside the container and the average moisture content are also controlled in order to obtain robust results (CV < 15%). According to one embodiment of the invention, an indexing conveyor is used and the method comprises the step of placing the open top container on the indexing conveyor belt and passing the container under the UV light source such that the vertical axis between the lamp and the container bisects the container into two symmetrical halves. This is explained further in Figure 3.

It will be understood that ideally the mushrooms receive 1, 2, 3, 4, or 5 pulses of UV irradiation.

According to another embodiment of the invention, the process comprises the step of pre-selecting one day post harvested mushrooms. In this manner, the pre-seiected mushrooms once harvested are protected from light exposure and stored at 4°C.

According to a preferred embodiment of the invention, the process comprises the additional measuring step of measuring the dry matter content of the mushrooms wherein mushrooms are selected with a dry matter content from 6 to 11 %. Dry matter is measured using British Standard method BS EN 14346:2006.

According to another preferred embodiment of the invention mushrooms are selected with a diameter from 20 to 120 mm, preferably from 40 mm to 120 mm, even more preferably from 40 mm to 55 mm. ideally, the mushrooms have a diameter not less than 40 mm.

According to yet another preferred embodiment of the invention mushrooms are selected with a weight from approximately 120 g to 800 g, preferably approximately 200 g.

According to another preferred embodiment of the invention mushrooms are selected with a density of from approximately 0.35 to 0.5 g/cm3.

According to another preferred embodiment of the invention mushrooms are selected with a surface area to volume ratio (SA/V) from approximately 0.5 to 1.25 cm'1, preferably from 1.09 to 1.46 cm'1.

According to another embodiment of the invention the distance from the container to the UV light source is from 10 to 27 cm.

The process of the invention may also comprise the step of packaging the container with mushrooms after UV irradiation.

Ideally, the mushroom is a brown or white mushroom of the Agaricus genus although mushrooms of any type may be used.

Further details on aspects of the process of the invention follow.

Mushroom Production The commercial production of Agaricus bisporus is a multi-step process. As Agaricus bisporus is a saprophytic type of fungi it is commercially grown in composted substrate which provides nutrients for the mushrooms to grow.

The commercial production of Agaricus bisporus is basically carried out in six main steps. These six steps include Phase I composting, Phase II composting, Phase III (spawning), casing, pinning and harvesting.

PHASE I COMPOSTING is the first step involved in mushroom farming. This first step is to create compost with sufficient nutrients to grow mushrooms and at the same time, provide little or no nutrition for other fungi or competitor organisms. This process involves much higher temperatures than those required in Phase il composting.

PHASE II COMPOSTING is the second step in mushroom farming, after Phase I composting is complete and the compost has been filled into containers or into a structure. Phase II composting has two main purposes: formal conditioning of compost so it becomes mushroom specific (absence of ammonia and readily available carbohydrates) and pasteurization. This step is performed under controlled environmental conditions and at much lower temperatures than Phase I composting.

PHASE III COMPOSTING (SPAWNING) is the placement of a spawn (rye, wheat, millet, or sorghum grain cooked with water and chalk, then sterilized. Mycelium is added and allowed to grow for 10-17 days) into compost prepared in Phase I and Phase II composting. By whatever method chosen or available, the importance of cleanliness, applying and mixing thoroughly cannot be over emphasized.

The casing operation is the fourth step in mushroom farming and is a top dressing placed directly on spawn- run compost from 14 to 21 days after the spawning operation.

The fifth step in mushroom farming and is initiated when rhizomorphs form in the casing and then emerge at the surface of the casing. There are two important steps involved in causing the mycelium to go from the vegetative stage to the fruiting stage. The two steps are to lower the air temperature approximately 10°C and introduction of outside fresh air to purge the CO2 from the surface of the casing. At the time of pinning, a final watering is sometimes added to protect against drying out of the casing layer due to the introduction of fresh air.

The sixth step in mushroom fanning begins 16-20 days following casing when the first mushrooms are harvested. Mushrooms are generally harvested (picked) for 3 to 5 days, followed by several days when no mature mushrooms are present. This cycle is repeated in a rhythmic fashion for the duration of the crop and is also called a flush (van Griensven, 1989).

The production process of the vitamin D2 enhanced Agaricus bisporus adds an additional step after harvesting, on which samples are placed in a packaging container and subject to pulsed UV-light.

Duration of harvesting can lead extended for 35-42 days, depending on the crop. Mature mushrooms are then hand-picked according to the specification requirement (button mushrooms for small diameter, closed cup mushrooms for medium diameter and portobello mushrooms for large diameter).

UV treatment Once mushrooms are picked and packed, they are ready for to be processed according to the method of the present invention.

This method of the present invention is ideally applied to day 1 mushrooms (i.e. mushrooms one day after being harvested).

The mushrooms are packed in a single layer in an open top container, with pileus or mushroom cap facing upwards. The gill is ideally adjacent to the inside base of the open top container to minimise exposure to the UV irradiation.

The mushrooms are ideally subject to broad spectrum pulsed light in the spectrum from 190-1100 nm.

Ideally, the light is any commercial UV spectrum light source, ideally a commercial scale pulsed light system. We have used Xenon LiteMark-XL High Intensity Pulse UV Light Detection System combined with a PicoLog 1000 Series Voltage logger and a Steripulse-XL-3000 pulsed light sterilization system (Xenon Corporation, MA, USA).

Although UV-B light has been claimed to be responsible of the conversion from ergosterol to ergocalciferol in mushrooms, this has not been proven. In addition, we have found that the Vitamin D levels can be precisely obtained using a lamp emitting with a large range of wavelengths.

Depending on the distance set at time of operation the broadband energy applied ranged from 0.6 to 15 mJ/cm2, optionally 0.6 to 5 mJ/cm2' preferably from 0.68 to 10 mJ/cm2 more preferably from 0.8 to 5 mJ/cm2, even more preferably from 1 to 4 mJ/cm2, still more preferably from 2.5 to 3.5 to 5 mJ/cm2.

The value of the dose changes significantly depending on the type of mushroom (classified by surface to volume ratio), the location of sample and resultant distance to the UV light source/lamp. The flash lamp unit is set to expend a calculated 526 Joules of energy per pulse.

In one embodiment of the invention, three pulses per second were applied to each pack. In order to avoid bias in the process, the mushrooms are protected from light at all times before and after treatment. Vitamin D analysis was carried out using a modified method described below which is based upon a British Standard (BS EN 12821:2009) which was validated against an accredited laboratory.

Once the mushrooms are treated under UV-light, are packed and ready for dispatch.

The present invention will be described with respect to the following non-limiting figures and examples.

Figure 1 is a flowchart showing the general steps in the process of the invention.

Figure 2 is an outline drawing of the UV treatment machine. This figure illustrates how the configuration of the light source and the positioning of the product allow for a repeatable process with consistent results from batch to batch.

Figure 3 is an outline drawing showing the positioning of the UV lamp relative to the mushrooms.

Figure 4 is a contour plot with Vitamin D2 contents that can be obtained from closed cup mushrooms at combination of distance to the lamp and number of pulses.

As shown in Figure 1 (a), the mushroom product of specific grade (density, surface area to mass ratio) after harvesting at an ambient temperature of 17-19°C are placed into punnets, stalk downwards. In step (b) the punnets are transported to cold store and chilled to 4°C, before treatment, to maintain grading. Step (c) shows the punnets being processed through the UV treatment machine (see Figure 2) before being sealed and labelled (not shown).

Figure 2 provides an outline figure of the UV treatment machine showing the Guide rails (a), Indexing conveyor (b), Conveyor flights (c), Punnet of product (d), Area of light spread (e), Angle of light spread (f), Light source (g), and Sealed chamber (h).

In use, the light source (g) is set at a known fixed height above the product and delivers a set number of fixed energy pulses of UV light inside the sealed chamber (h). Light emitted from source (g) spreads at a fixed angle (f).

In order to ensure consistent positioning under the light source (g), the mushrooms are placed stalk facing downwards in the punnets (d). The punnets are positioned between guide rails (a) and conveyor flights (c). This combined with the use of an indexing conveyor belt (b) and positioning sensors ensures consistent positioning for each batch.

As shown in Figure 3, the vertical axis between the lamp and the container cuts the container in two symmetrical halves. It is important that the mushrooms are positioned in such a manner that each mushroom is a relative equal distance to that vertical axis. This is important to maintain a relatively similar intensity of UV over the surface of the mushrooms.

EXAMPLES Example 1 - Production of Vitamin D2 enhanced Heirloom Flush 2 closed cup mushrooms Materials Acronym Definition HL Heirloom CC F2 Closed Cup Flush 2 RDA Recommended Daily Allowance UV Ultraviolet Xenon UV-C type lamp Flush 2 Heirloom closed cup (HL F2 CC) mushrooms - the experiments were conducted with Flush 2 Heirloom closed cup mushrooms with a specification of 50 mm with a surface/volume ratio of around 1.27 cm1. All mushroom samples were protected from extraneous light exposure throughout the experiments. After harvesting the harvester was instructed to cover the samples with brown paper. These samples were all sourced from Kernans farm (Armagh, NI, United Kingdom) and delivered to Monaghan Mushrooms, Tyholland. Upon delivery the samples were stored at 4°C until exposure to pulsed UV light.

Method Brown Agaricus bisporus (HL) closed cup mushrooms were harvested and placed in 200 g lots into polypropylene containers (punnets). Six mushrooms were arranged carefully cap side up in to the punnets as to not over expose the gills.

The pack of mushrooms are placed on a conveyor belt, as the mushroom pack passes through a detector, the hood containing the lamp lowers and encloses the pack of mushrooms and triggers the pulsed UV-light process.

All samples were placed in the pulsed UV system at a distance of 11.7, 16.5 and 21.5 cm from the UV lamp and were exposed for 1, 3 and 5 pulses. All the above distance and pulses were tested to establish the best pulse regime to increase vitamin D2 content in HL F2 CC mushrooms to a value of circa 200 % of the RDA. The product was placed on the system and the samples were all exposed to pulsed UV light individually. Light intensity was measured with a broadband silicone detector SED033/QNDS for the energy range between 200-1100 nm. Measurements were taken at different positions to confirm values were around 3.2 mJ/cm2 for a fixed distance between the lamp and the detector.

The samples were exposed to pulsed UV light on day one of shelf-life and wrapped in aluminium foil immediately after treatment, to eliminate excess exposure to light. The samples were stored at 4°C until analysis.

Analyses were carried on day 8 of shelf life. Analyses were carried out on day 8 to determine the vitamin D2 content in the mushroom samples at the end of shelf life. Previous research has indicated that there is no significant difference (degradation) in Vitamin D content from the start to the end of shelf life.

Treated samples were sent to an accredited food and pharmaceutical laboratory for vitamin D2 analysis. Vitamin D2 values of fresh mushrooms are presented in pg/100g fresh weight.

Results and Discussion After exposure to increasing number of pulsed UV light there was an increase in the vitamin D2 content in all the brown Agaricus bisporus mushrooms tested. With a reduced distance of the UV lamp to the product and an increasing number of pulses, higher amounts of vitamin D2 was produced.

Fresh HL F2 CC mushrooms showed an increase from 0 pg/100g FW to 3 pg/100g FW at a distance of 16.5 cm with 3 pulses. After 5 pulses at a distance of 11.7 cm the vitamin D2 content had increased to 10 pg/100g FW. Results indicated that when the base of the product is placed at a distance of 11.7 cm from the UV lamp with 5 pulses enough vitamin D2 is produced to be in the range of circa 200% RDA (10 pg/100g FW). These results are obtained for HL F2 CC mushrooms, the orientation of these mushrooms was considered carefully when being placed on the UV system as alteration will affect the results. The specification of mushroom was strictly followed during these experiments.

HL F2 CC mushrooms were also analysed on day 8 of shelf life to determine the vitamin D2 content at the end of shelf life. Two production runs were completed over 2 consecutive days. The base of product was placed at a distance of 11.7 cm from the lamp and 5 pulses were emitted during these production runs.

Table 2. Vitamin D2 results obtained from production run 1 and 2 on day 8 of shelf life.

Production run 1 Production run 2 (ug /100 g FW) (ug/100g FW) Replicate 1 7.08 6.98 Replicate 2 7.28 6.07 Replicate 3 8.73 5.33 Replicate 4 7.53 6.93 Replicate 5 6.75 7.05 Average 7.47 6.47 Standard Deviation 0.76 0.75 Coefficient of variation (%) 10.14 11.63 Both production run 1 and 2 were both analysed on day 8 of shelf life to establish the vitamin D content in HL F2 CC mushrooms at the end of shelf life. Similar results were obtained from both 10 production runs. Statistically there is no significant difference in in the results achieved (p>0.05). The results obtained from the two production runs indicate that there is little variability from the 5 samples on any one day as the vitamin D2 content is quite consistent. The coefficient of variation (%CV) from production run 1 and 2 are very similar which shows that there is no variability from inter day analysis. Coefficient of variation is the ratio between the standard deviation and the average and expressed as a percentage. It gives an indication of the variability of the data set. When the coefficient of variation is low (below 20%) is considered a robust, repeatable process.

This study demonstrates that after a short exposure time of 2 seconds, the vitamin D2 content in HL F2 CC mushrooms can be increase to circa 200% at the start of shelf life and upwards of 150% the RDA per serving at the end of shelf life.

Conclusion It can be concluded that optimum results are achieved when the UV lamp is at a distance of 11.7 cm from the product and 5 pulses of UV light are emitted from the lamp. The UV-C type bulb was very effective in converting ergosterol to ergocalciferol in Heirloom flush 2 closed cup mushrooms.

EXAMPLE 2 - Vitamin D2 content in Agaricus bisporus mushrooms Large flat Agaricus bisporus mushrooms, with a surface to volume ratio of around 0.86 cm'1 (this represented an average exposed surface value of 100 cm2) were treated according to the invention at different combinations of distance from the lamp and number of pulses.

Materials Xenon UV-C type lamp Agaricus bisporus mushrooms - the experiments were conducted with brown different sizes of mushrooms. Harvesting of the mushrooms was carried out with for specification of 85 mm of cap diameter for large (portobellos). After harvesting the harvester was instructed to cover the samples with brown paper. These samples were all sourced from Kernans farm (Armagh, NI, United Kingdom) and delivered to Monaghan Mushrooms, Tyholland. Upon delivery the samples were stored at 4°C until exposure to pulsed UV light.

The exposed surface and surface to volume ratio was calculated as indicated in previous examples Mushroom samples with surface to volume ratios of 0.6 to 1.0 were selected, placed in a punnet for subsequent UV-treatment.

Method The Agaricus bisporus (HL) mushrooms were harvested and placed in 200 g lots into polypropylene containers (punnets).

The pack of mushrooms are placed on a conveyor belt, as the mushroom pack passes through a detector, the hood containing the lamp lowers and encloses the pack of mushrooms and triggers the pulsed UV-light process.

The mushroom samples were exposed to an increasing number of pulses at specific distances and vitamin D level then analysed. The samples were protected from sunlight and immediately stored at 4°C after the treatment until analysis.

Treated samples were analyzed for vitamin D2 analysis. Vitamin D2 values of fresh mushrooms are presented in pg/100g fresh weight.

Results and Discussion Table 3. Vitamin D2 results obtained from portobello mushrooms affixed distance and increasing 5 number of pulses.

Sample Distance Number of pulses Energy Integrated Vitamin D content Average Vitamin D content Stdev (cm) (mJ/cm2) (ug/100 g FW) Portobello 1 12 1 0.68 7.19 0.75 Portobello 2 12 2 1.36 21.02 3.16 Portobello 3 12 3 2.04 25.12 1.19 Results from Table 3 show that for a specific distance between lamps and mushrooms the vitamin D content increased exponentially with the number of pulses. Above a given number of pulses the vitamin D content reached a plateau and did not increased significantly when the number of pulses was increased.

Table 4: Vitamin D2 results obtained from portobello mushrooms at fixed number of pulses and increasing distance.

Vitamin D content Sample Distance Number of pulses Energy Integrated* Average Stdev (cm) (mJ/cm2) (ug/100 g fresh weight) Portobello 4 8 1 0.68 12.84 0.33 Portobello 5 12 1 0.68 7.38 1.25 Portobello 6 35 1 0.68 1.27 0.7 Portobello 7 5 2 1.36 51.22 1.52 Portobello 8 12 2 1.36 21.02 3.16 Portobello 9 17 2 1.36 13.14 2.34 * integrated energy values of the lamp at a fixed distance gives an indication that energy at 0.68 mJ/cm2 of one single pulse has an impact in vitamin D content depending on distance (8/12/35cm).

Results from Table 4 show that when a fixed set of number of pulses was used the content of vitamin D in mushrooms decreased when distance between source tamp and surface of mushrooms.

Portobello 6 shows that where the distance is greater than 5 to 30cm that the Vitamin D content is less than 5 to 40 ug/100 g.

Conclusion The results show that the distance to the lamp and the number of pulses can be modified for a specific type of mushrooms defined by surface exposed and/or surface to volume ratio) in order to obtain very precise and controlled values of vitamin D not previously possible.

EXAMPLE 3 - Optimization of Vitamin D2 content in closed cup mushrooms using Response Surface Methodology.

Materials Acronym Definition RDA Recommended Daily Allowance UV Ultraviolet Xenon UV-C type lamp Agaricus bisporus mushrooms - the experiments were conducted with brown different sizes of mushrooms. Harvesting of the mushrooms was carried out mushrooms with a cap diameter of approximately 30 mm (S/V ratio: 2.09 cm'1), 45 mm (S/V ratio: 1.27 cm'1) and 85 mm (S/V ratio: 0.86 cm'1) of cap diameter for small (button mushrooms) medium (closed cup mushrooms) and large (portobello mushrooms) mushrooms respectively. After harvesting the harvester was instructed to cover the samples with brown paper. These samples were all sourced from Kernans farm (Armagh, NI, United Kingdom) and delivered to Monaghan Mushrooms, Tyholland. Upon delivery the samples were stored at 4°C until exposure to pulsed UV light. RSM Statistical program Design Expert version 9.0.3.1 developed by Stat-Ease Incorporated (Minneapolis, USA) The surface and surface to volume ratio was calculated using for this purpose the general mathematical ellipsoid formula approximation. Those samples with surface to volume ratios of 1.0 to 1.5 cm1 were selected for their RSM optimisation.

Method The Agaricus bisporus (HL) mushrooms were harvested and placed in 200g lots into polypropylene containers (punnets).

The pack of mushrooms are placed on a conveyor belt, as the mushroom pack passes through a detector, the hood containing the lamp lowers and encloses the pack of mushrooms and triggers the pulsed UV-light process.

A central composite design was carried out for 2 numeric factors (distance from the lamp and number of pulses). Each factor ranged from maximum to minimum values. Three centre points were chosen to determine variability of the method. The experimental design returned the number of distance and number of pulses combinations to run to construct the model. The mushroom samples were exposed to these combinations pulsed UV light and vitamin D level was used as response factor for the mathematical model. The samples were wrapped in aluminium foil immediately after treatment. The samples were stored at 4°C until analysis.

Treated samples were sent to an accredited food and pharmaceutical laboratory for vitamin D2 analysis. Vitamin D2 values of fresh mushrooms are presented in pg/100g fresh weight. The values from the accredited laboratory were then used to construct the mathematical model. The mathematical model was assessed statistically and is able to predict successfully vitamin D contents for a specific mushroom type for known values of distance and number of pulses.

Results and Discussion The results are shown in Figure 4 which is a contour plot with Vitamin D2 contents that can be obtained from closed cup mushrooms at combination of various distances to the lamp and number of pulses.

The model F-value of 6.55 implied the model was significant (is a good model to predict vitamin D levels). There was only a 2.03% chance that the F-value this large could occur due to noise. The Lack of Fit F-value of 4.19 implied the Lack of Fit is not significant relative to the pure error. There was a 13.50% chance that a Lack of Fit F-value this large could occur due to noise. Non-significant lack of fit is generally considered as good as it gives an idea if the mathematical model is correct.

EXAMPLE 4 - Comparison of Vitamin D2 content in wild and UV treated A. bisporus samples Levels of vitamin D in wild Agaricus mushrooms have been confirmed by HPLC analysis based British Standard BS EN 12821:2009. Wild Agaricus bisporus were collected in around the Christchurch area in New Zealand equivalent latitude coordinates to southern France / Northern Italy. The vitamin D levels in wild mushrooms can be as high as 31.22 or as low as 1.37 pg per 100 g. Vitamin D content in mushrooms treated with UV light is more consistent than the vitamin D contents in wild mushroom (variation of 9.7 % versus 144 % respectively).

Vitamin D mushrooms produced by the present method fall within this range between 6.75 and 8.73 pg per 100g. Vitamin D content in mushrooms treated with UV light is more consistent than the vitamin D contents in wild mushroom (variation of 9.7 % versus 144 % respectively). No significant difference (P>0.05) in vitamin D content between mushrooms treated with UV-light and those harvested from wild was detected.

Table 5. Summary of the results, average, standard deviation and coefficient of variation (% CV) of vitamin D content (in pg/100g fresh weight) wild and UV treated A. bisporus samples.

Sample Wild A. bisporus UV treated A. bisporus 1 6.17 7.08 2 1.7 7.28 3 31.22 8.73 4 4.57 7.53 5 2.95 6.75 6 1.37 6.89 Average 8 7.38 Standard deviation 11.52 0.72 Coefficient variation 144 9.74 These results show that human exposure to vitamin D by consuming wild Agaricus bisporus is in fact probable and also that this exposure is not controlled and high levels of vitamin D might be attained in wild mushrooms (>600 % of the EU RDA). The present method provides mushrooms with similar average Vitamin D levels as wild mushrooms; and in a more precise manner.

Conclusion Considering that HPLC method used to test Vitamin D content has an experimental error of around 10% and that the correlation coefficient between predicted and analysed data is R2 = 0.86, it can be concluded that vitamin D content in mushrooms can be optimized by firstly selecting the type of mushroom defined by diameter and subsequently surface to volume ratio as defined in the claims and then exposing mushrooms to different combinations of distance and number of pulses within the ranges disclosed in the claims and using RSM to optimize and refine the process.

REFERENCES Mattila P, Piironen VI, Uusi-Rauva EJ, Koivistoinen PE. Vitamin D contents in edible mushrooms. J Agricultural & Food Chemistry 1994; 42: 2449-2453 Teichmann, A., Dutta, P. C., Staffas, A., & Jagerstad, M. (2007). Sterol and vitamin D2 concentrations in cultivated and wild grown mushrooms: Effects of UV irradiation. LWT - Food Science and Technology, 40(5), 815-822. doi:10.1016/j.lwt,2006.04.003 van Griensven, L J L D, The Cultivation of Mushrooms, ISBN 0951395904, 515 pages The invention is not limited to the embodiment(s) described herein but can be amended or modified without departing from the scope of the present invention.

Claims (13)

1. A process for increasing the Vitamin D content of mushrooms to approximately 5 to 40 pg Vitamin D per 100 gram comprising the steps of pre-selecting mushrooms from flush 1, flush 2 or flush 3; measuring the diameter and weight of the mushrooms; calculating surface area to volume ratio, and density of the mushrooms; selecting mushrooms with a density from approximately 0.25 to 0.5 g/cm 3 and a surface area to volume ratio (sa/vol) from approximately 0.5 to 2.5 cm' 1 ; placing a single layer of mushrooms in an open top container wherein the mushrooms are placed with the mushroom cap facing upwards such that the gill is adjacent to the inside base of the open top container; passing the open top container with mushroom cap facing upwards under a UV light source wherein the distance from the inside base of the open top container to the UV light source is from approximately 5 to 30 cm and the mushrooms receive from 1 to 5 pulses of UV irradiation to result in the mushrooms receiving from approximately 0.6 to 15 mJ/cm 2 of UV irradiation.

2. The process according to claim 1 wherein an indexing conveyor is used and the method comprises the step of placing the open top container on the indexing conveyor belt and passing the container under the UV light source such that the vertical axis between the lamp and the container bisects the container into two symmetrical halves.

3. The process according to claim 1 or claim 2 wherein the mushrooms receive 1, 2, 3, 4, or 5 pulses of UV irradiation.

4. The process according to any of the preceding claims comprising the step of pre-selecting one day post harvested mushrooms.

5. The process according to any of the preceding claims wherein the pre-selected mushrooms once harvested are protected from light exposure and stored at 4°C.

6. The process according to any of the preceding claims comprising the additional measuring step of measuring the dry matter content of the mushrooms wherein mushrooms are selected with a dry matter content from approximately 6 to 11%.

7. The process according to any of the preceding claims wherein mushrooms are selected with a diameter from approximately 20 to 120 mm, preferably less than 55 mm. 5

8. The process according to any of the preceding claims wherein mushrooms are selected with a weight from approximately 120 g to 800 g, preferably approximately 200 g.

9. The process according to any of the preceding claims wherein mushrooms are selected with a density from approximately 0.35 to 0.4 g/cm 3 .

10. The process according to any of the preceding claims wherein mushrooms are selected with a surface area to volume ratio (sa/vol) from approximately 1.09 to 1.46 cm' 1 .

11. The process according to any of the preceding claims wherein the distance from the 15 container to the UV light source is from approximately 10 to 27 cm.

12. The process according to any of the preceding claims comprising the step of packaging the container with mushrooms after UV irradiation. 20

13. The process according to any of the preceding claims wherein the mushroom is a brown or white mushroom of the Agaricus genus.