JP2019068843A - Yeast cell wall containing vitamin d2, use of the same, and method for producing the same - Google Patents

Abstract

To provide a yeast cell wall containing vitamin D2.SOLUTION: A yeast cell wall containing vitamin D2 is obtained by adding vitamin D2, or subjecting a yeast cell wall fraction to UV light, or subjecting yeast to UV light and then autodigestion and/or hydrolysis and separation.SELECTED DRAWING: None

Description

Contents of disclosure

Field of the Invention
The present application relates to an adjunct source of vitamin D applied to humans and animals. More specifically, the present application relates to yeast cell walls comprising vitamin D2, its use, and its method of production.

BACKGROUND OF THE INVENTION
Yeasts such as Saccharomyces are known to have high nutritional value, particularly as a source of vitamin B. For example, brewer's yeast has been marketed as a human supplement for many years. Other yeasts such as Torula, Candida, or Klyuveromyces have also been used as growth factor and vitamin supplements for human use and / or for animal feed. Recently, there has been a growing interest in vitamin D-containing yeasts, which are used to fortify foods used by humans and animals. However, yeast does not contain vitamin D, but contains a unique sterol called ergosterol, which is a provitamin D2 sterol. Yeast containing vitamin D are usually obtained by treating the yeast with a UV source.

[Summary of the invention]
According to one aspect of the present invention, there is provided a yeast cell wall comprising vitamin D2.

  In one aspect of the yeast cell wall, vitamin D2 is added vitamin D2.

  In another aspect of the yeast cell wall, vitamin D2 is obtained by treating the yeast or a fraction of the yeast cell wall with a UV source.

  In a further aspect of the yeast cell wall, the yeast cell wall is UV treated.

  In one aspect of a yeast cell wall comprising vitamin D2, the yeast cell wall is produced from Saccharomyces or any non-Saccharomyces yeast.

  In another aspect, the non-Saccharomyces yeast is selected from the group consisting of Candida sp., Hansenopsis sp., Hansenula sp., Kleberomyces sp., Methynicobia sp., Pichia sp., Starmerella sp.

  In the UV-treated yeast cell wall aspect comprising vitamin D2, the UV-treated yeast cell wall comprises β-glucan.

  In the embodiment of the UV-treated yeast cell wall comprising vitamin D2, the β-glucan content is at least 75% of the β-glucan content of the yeast cell wall not treated with UV light, the β of the yeast cell wall not treated with UV light At least 80% of the glucan content, at least 85% of the β-glucan content of the yeast cell wall not treated with UV light, or at least 90% of the β-glucan content of the yeast cell wall not treated with UV light.

  According to another aspect of the invention, the yeast cell wall or the UV-treated yeast cell wall as defined above is used as a nutraceutical for nutrition by animals or humans.

  According to a further aspect of the invention, the yeast cell wall or the UV-treated yeast cell wall as defined above may be baked goods, functional foods, dietary foods, nutritional foods, fermented beverages or non-fermented It is provided for use as an additive for beverages.

  According to a further aspect of the present invention there is provided a baked good manufactured using yeast cell walls or UV treated yeast cell walls as defined above.

  In one aspect of the baked good, the baked good is a bread, a cracker, sport bars, or a biscuit.

  According to another aspect of the present invention, there is provided a composition comprising an animal feed and a UV-treated yeast cell wall comprising vitamin D2.

  In one aspect of this composition, the composition is used in the diet of laying hens to increase vitamin D2 content in egg yolk.

  According to a further aspect of the present invention there is provided a method of increasing vitamin D2 content in egg yolk, the method comprising laying a composition comprising a feed of laying hens and a UV-treated yeast cell wall comprising vitamin D2. Including giving to.

  According to another aspect of the present invention, there is provided a method of preparing a UV-treated yeast cell wall comprising vitamin D2, the method comprising the steps of supplying yeast and yeast to obtain autodigestion and / or hydrolysis yeast. And / or hydrolyzing the yeast, separating the autolysed and / or hydrolyzed yeast to provide a yeast cell wall fraction and a yeast extract, UV treated yeast cell wall containing vitamin D2 Treating the yeast cell mural fraction with UV light to obtain.

  In some embodiments of this method, the vitamin D2 content is at least 100 times higher than the yeast cell wall not treated with UV light, or at least 200 times higher than the yeast cell wall not treated with UV light, or is treated with UV light Yeast at least 300 times higher than yeast cell walls, at least 400 times higher than yeast cell walls not treated with UV light, or at least 500 times higher than yeast cell walls not treated with UV light, or yeast not treated with UV light At least 1000 times higher than the cell wall, at least 5000 times higher than the yeast cell wall not treated with UV light, or at least 10000 times higher than the yeast cell wall not treated with UV light.

  According to a further aspect of the present invention there is provided a method of preparing a UV-treated yeast cell wall comprising vitamin D, the method comprising the steps of feeding the yeast and obtaining the UV-treated yeast to obtain a vitamin D2. Treating with UV light, and auto-digesting and / or hydrolyzing UV-treated yeast containing vitamin D2 to obtain UV-treated autolysed and / or hydrolyzed yeast, and comprising vitamin D2 Separating the UV-treated autolysed and / or hydrolyzed yeast so as to provide a UV-treated yeast cell wall fraction and a UV-treated yeast extract.

  In an embodiment of this method, the vitamin D2 content in the UV-treated yeast cell wall is at least 1.25 times, preferably at least 1.5 times, more preferably at least 1.5 times the content of vitamin D2 in the UV-treated yeast containing vitamin D2. Preferably at least 1.75 times, most preferably at least 2 times.

[Detailed description]
Vitamin D plays an important role in both human and animal health. Humans can produce vitamin D, especially vitamin D3, when exposed to UV radiation from sunlight. Also, vitamin D can be obtained through the diet, especially vitamin D fortified milk. These vitamin D sources are becoming insufficient to provide the vitamin D levels needed for good health, especially in adults, who spend less time in direct sunlight and consume less milk. There is.

  For example, nutritious bread and cereals have become a supplemental source of vitamin D in the diet. However, commercially available vitamin D3, used as an additive, has been separated from animal sources by extraction and purification steps and is an unacceptable additive for at least a portion of the population.

  Acceptable alternative sources of vitamin D can be found in yeast. However, yeast does not contain vitamin D and contains its own sterol, ergosterol, which is a provitamin D2 sterol. For example, in bread made with yeast containing ergosterol, one of the methods known for increasing vitamin D requires that bakers treat the bread with UV light. Other methods known to increase vitamin D generally involve UV treatment of active yeast at low temperatures to avoid yeast autolysis, which results in yeast viability, enzyme activity, and vitamin D content. Keep

  It has been observed that ergosterol is mainly divided into yeast cell wall by-products after the yeast autolysis and / or hydrolysis and separation processes. Ergosterol has been found to remain stable under the autolysis and / or hydrolysis and separation processes of yeast and to retain most of its ability to be converted to vitamin D2. As a result, more ergosterol content that can be converted to vitamin D2 with UV light can be obtained in the yeast cell wall by-product. This has been found to be advantageous for providing humans and animals with vitamin D levels necessary for good health. It has also been found to be advantageous to provide humans and animals with a cheap supplemental source of vitamin D not isolated from animal sources. Furthermore, it has been found to be advantageous for providing humans and animals with added value food and feed additives and added value baked goods.

  The present application relates to a yeast cell wall comprising vitamin D2, its use and a method of producing it.

  It will be understood that the term "yeast cell wall" as used herein refers to a by-product obtained by autolysis and / or hydrolysis of yeast. In this process, insoluble cell walls are separated from soluble components (yeast extract). The yeast cell wall is mainly composed of β-glucan, mannoproteins and proteins. As mentioned above, ergosterol is mainly divided into yeast cell walls after autolysis and / or hydrolysis of yeast.

  The terms "UV-treated" or "treated with UV light" as used herein, were yeast cell walls or yeast exposed to UV light for the purpose of increasing the vitamin D2 content therein It will be understood to refer to the process to be exposed or exposed.

  It is understood that the term "UV-treated yeast cell wall comprising vitamin D" as used herein refers to a yeast cell wall as described above, wherein the content of vitamin D2 is increased upon treatment with UV light. Will be done.

  Reference will now be made to the embodiments shown in the drawings and described herein. It is thereby understood that no limitation of the scope of the disclosure is intended. It is further understood that the disclosure includes all changes and modifications to the illustrated embodiments, and further applications of the principles of the disclosure as would normally occur to one skilled in the art to which this disclosure belongs.

  In one embodiment, a yeast cell wall comprising vitamin D2 is provided. Vitamin D2 may be added vitamin D2 (added vitamin D2). The added vitamin D2 may be obtained from any suitable source for the present application and mixed with the yeast cell wall.

  In another embodiment, the cell wall comprising vitamin D2 is a UV treated yeast cell wall comprising vitamin D2. Also provided is a method of obtaining a UV-treated yeast cell wall comprising vitamin D2.

  Referring to FIG. 3, a method 10 of preparing a UV-treated yeast cell wall comprising vitamin D2 according to the present application is shown. At step 20, the method 10 comprises providing a yeast. The yeast may be a yeast from Saccharomyces, or any non-Saccharomyces yeast. The non-saccharomyces yeast is selected from the group consisting of Candida species, Hansenia spora species, Hansenula species, Kleberomyces species, methynikobia species, Pichia species, Starmelera species, and Truluspora species. Preferably, the yeast is Saccharomyces cerevisiae or Cyberlindnera jadinii (Torula yeast).

  In step 22, the yeast of step 20 is subjected to autolysis and / or hydrolysis according to methods known to those skilled in the art in order to obtain autolyzed and / or hydrolyzed yeast. In one embodiment, at least one enzyme may be used for yeast hydrolysis. Preferably, the at least one enzyme is papain. It is understood that other suitable enzymes or enzyme mixtures intended to hydrolyze yeast can be used. In one embodiment, autolysis and / or hydrolysis is carried out at a temperature of 50-65 ° C., preferably 55-60 ° C., for example, 20-28 hours, preferentially 22-24 hours. Over a period of time.

  The autolysed and / or hydrolyzed yeast of step 22 may be inactivated by heat treatment at a temperature of 75-85 ° C. for 15-30 minutes, in a manner known to those skilled in the art.

  In step 24, the autodigested and / or hydrolyzed yeasts of step 22 are separated to yield a yeast cell wall fraction and a yeast extract. More specifically, the soluble fraction (yeast extract) is separated from the insoluble portion (yeast cell wall fraction) according to methods known to those skilled in the art, for example by using a centrifuge. It is understood that any other suitable method of separating the soluble fraction from the insoluble fraction may be used. However, yeast extracts that do not form part of the present application may be used in other suitable applications, such as, for example, the production of food, wine, beer and fuel ethanol.

  In step 26, the liquid yeast cell wall fraction obtained from step 24 is treated with UV light to increase the content of vitamin D2 therein to produce a UV treated yeast cell wall containing vitamin D2. In order to increase the content of vitamin D2 in the yeast cell wall, the yeast cell wall fraction of step 24 is a UV photo-bioreactor system with agitation as shown in FIG. 1 or FIG. 2 (UV photo-bioreactor system with agitation) Can be treated with UV light. On a small scale, eg laboratory scale, yeast cell wall fraction can be treated with UV light in the surface UV photo-bioreactor of FIG. 2 for about 2 to 6 hours, preferably about 4 hours . The yeast cell mural fraction obtained from step 24 may be treated with UV light at a temperature of about 4 ° C to about 60 ° C, preferably about 50 ° C. In another embodiment, the UV light may be located at a distance of 2.5 to 10 cm from the surface of the yeast cell wall to be treated.

  Alternatively, on a large scale, the yeast cell mural fraction obtained from step 24 may be exposed to UV light in a submerged photo-bioreactor as shown in FIG. 2 for a longer period of time, eg, 24-120 hours. It can be processed. It is understood that processing of the yeast cell wall with a UV light requires a shorter or longer period, as required. In large scale, yeast cell walls may be treated with UV light at a temperature of about 4 ° C to about 60 ° C, preferably at a temperature of about 4 ° C. In one embodiment, UV light can be submerged in the photobioreactor.

  In one embodiment, the wavelength of UV light may be in the range of 200-400 nm, preferably the UVB band, more specifically 302 nm, or preferably the UVC band, more specifically 254 nm.

  The corresponding UV-treated liquid yeast cell wall containing vitamin D2 from step 26 can be further concentrated and spray-dried to obtain a powder of UV-treated yeast cell wall containing vitamin D2. It is understood that other suitable methods, such as, for example, roller drying, can be used for the purpose of obtaining dried UV-treated yeast cell walls containing vitamin D2.

  In an embodiment of the method 10, the content of vitamin D2 in the UV-treated yeast cell wall as described above is at least 50 times, more preferably at least 100 times, at least 200 times as compared to the non-UV-treated yeast cell wall , At least 300 times, at least 400 times, at least 500 times, at least 1000 times, at least 5000 times, at least 10000 times.

  Referring to FIG. 4, an alternative method 110 of preparing a UV-treated yeast cell wall comprising vitamin D according to the present application is shown. At step 120, the method 110 includes providing a yeast. The yeast is as defined in the description of method 10.

  In step 122, the yeast of step 120 is treated with UV light to obtain UV-treated yeast comprising vitamin D2. The yeast is exposed to the same conditions of UV light, time and temperature as defined in the description of method 10.

  In step 124, the UV-treated yeast containing vitamin D2 obtained in step 122 is subjected to autolysis and / or hydrolysis to contain vitamin D2, according to methods known to those skilled in the art. Obtain hydrolyzed UV-treated yeast. In one embodiment, at least one enzyme may be used for hydrolysis of UV-treated yeast. Preferably, at least one enzyme is papain. It is understood that other suitable enzymes or enzyme mixtures may be used for the purpose of autolysing / hydrolyzing yeast. In one embodiment, autolysis / hydrolysis may be performed at a temperature of 50-65 ° C., preferentially 55-60 ° C., for example for a period of 20-28 hours, preferentially 22-24 hours. .

  The self-digested and / or hydrolyzed UV-treated yeast containing vitamin D2 obtained from step 124 is heat-treated at a temperature of 75-85 ° C. for 15-30 minutes in a manner known to those skilled in the art It can be inactivated.

  In step 126, the UV digested and / or hydrolyzed UV-treated yeast is separated to provide a UV-treated yeast cell mural fraction comprising vitamin D2 and a yeast extract. More specifically, the soluble fraction (yeast extract) is separated from the insoluble portion (UV-treated yeast cell mural fraction containing vitamin D2) according to the same appropriate method as defined in method 10. However, yeast extracts that do not form part of the present application may be used for other suitable applications, as defined in method 10.

  The corresponding UV-treated liquid yeast cell wall fraction containing vitamin D2 from step 126 can be further concentrated and spray-dried to obtain a powder of UV-treated yeast cell wall containing vitamin D2. It is understood that other suitable methods, such as, for example, roller drying, can be used to obtain dried UV-treated yeast cell walls containing vitamin D2.

  In an embodiment of the method 110, the percentage of vitamin D 2 in UV-treated commercial liquid yeast divided into yeast cell walls after the autolysis / hydrolysis and fractionation step is at least 75%, more preferably at least 80%. At least 85%, at least 90%, and at least 95%. Depending on the yield of the separation step, the vitamin D2 content in the UV-treated yeast cell wall is at least 1.25 times, preferably at least 1.25 times the vitamin D2 content in the UV-treated yeast comprising vitamin D2. 5 times, more preferably at least 1.75 times, most preferably at least 2 times.

  In one embodiment, the UV-treated yeast cell wall further comprises β-glucan, which is also known to have many health benefits for humans and animals. As shown in FIG. 5, the β-glucan content in the yeast cell wall, measured before and after UV light treatment at different wavelengths and temperatures, is very similar to each other. Before UV light treatment, the yeast cell wall has a β-glucan content of 29.17% by weight relative to the total weight of the yeast cell wall on a dry basis. With 254 nm UV light, yeast cell walls treated at 4 ° C. have a β-glucan content of 31.41% by weight relative to the total weight of the yeast cell walls on a dry basis. The yeast cell wall treated at 50 ° C. with 302 nm UV light has a β-glucan content of 30.38% by weight relative to the total weight of the yeast cell wall on a dry basis. With reference to these results, it can be concluded that UV-treated yeast cell walls containing vitamin D2 retain most of their β-glucan content after UV light treatment, regardless of the conditions. More specifically, UV-treated yeast cell walls comprising vitamin D2 are at least 75%, at least 80%, at least 85%, at least 90% of their β-glucan content relative to non-UV-treated yeast cell walls Or at least 95% can be retained.

  In one embodiment, a composition comprising animal feed and a UV-treated yeast cell wall comprising vitamin D2 is provided. The composition can further comprise β-glucan. This composition can be used in the diet of laying hens to increase vitamin D2 content in egg yolk. It is understood that the composition may be used in the diet of animals as a dietary supplement. In one embodiment, the animals may be cows, pigs, birds, horses, sheep, lapines, goats, dogs, and cats. Preferably, the birds are chickens, turkeys, ducks, geese, pheasants, quails, or companion birds.

  In another embodiment, there is provided a method of increasing vitamin D content in egg yolk, the method comprising: adding a composition comprising a feed of laying hens and a UV-treated yeast cell wall comprising vitamin D to a laying hens Including giving.

  In one embodiment, UV-treated yeast cell walls comprising vitamin D2 may be used as an additive for baked goods, functional foods, diet foods, nutraceuticals, fermented drinks or non-fermented drinks. The UV-treated yeast cell wall may further comprise β-glucan.

  In one embodiment, there is provided a baked good manufactured using UV-treated yeast cell wall comprising vitamin D2. The UV-treated yeast cell wall can further comprise β-glucan. Preferably, the baked good is a bread, a cracker, a stick for sports, or a biscuit.

  In one embodiment, there is provided a product comprising vitamin D2 for human nutrition, wherein the vitamin D2 content is comparable to the regulatory requirements of daily vitamin D intake from the diet.

  In one embodiment, UV-treated yeast cell walls can be used as a nutrient or vitamin source in fermented beverages and fermented foods.

〔Example〕
Instruments The photobioreactors of FIGS. 1 and 2 were used to assess the increase in vitamin D2 in the yeast cell wall by UV treatment. The bioreactor 1 contains a solution containing a volume V1 of yeast material 2 which is not in direct contact with a UV lamp 3 located at a distance of 5 cm from the solution surface (FIG. 1). Alternatively, the bioreactor 1 comprises a solution 2 of volume V2 greater than V1 and an ultraviolet lamp 3 is inserted into the solution with a quartz sleeve 4 (FIG. 2). A stirrer 5 is used to ensure solution homogenization during processing.

Example 1: Autolysis / Hydrolysis and Yeast Fractionation Yeast cell walls and yeast extracts were produced from Saccharomyces cerevisiae using an enzyme enhanced autolysis / hydrolysis process. Using a common commercial baker's yeast cream, the solid content of cream yeast was adjusted to 14.99% before fractionation experiments. Six liters of liquid yeast were hydrolysed in a Chemap fermenter for 22 hours at 55 ° C., pH 5.6, stirring 600 rpm. Papain (Promod 144 GL from Biocatalysis) was added at a rate of 0.5% based on the dry matter content of liquid yeast. After 22 hours of autolysis / hydrolysis, the hydrolyzate was inactivated by heat treatment at 85 ° C. for 30 minutes. After cooling the yeast hydrolyzate, separation was performed using a laboratory centrifuge (Sorvall Legend XTR, 10000 g from Thermo Scientific) to wash and collect the yeast cell wall fraction. The weight of the liquid cell wall and liquid yeast extract was determined. Dry gravimetric analysis (solid content) was performed on three samples to perform mass balance analysis: liquid yeast sample at the beginning of hydrolysis, hydrolyzed after heat inactivation treatment followed by hydrolysis Yeast sample, and final liquid yeast cell wall sample.

表１：通常のパン酵母の分別中の乾燥物質収支
（^＊：ラレマンド社（Lallemand）の酵母培養物コレクション） After separation, dry cell wall samples were prepared from liquid cell walls. To prepare the dried cell wall sample, the liquid cell wall was filtered or centrifuged and pressed to produce cell wall clumps. The mass was then dried in an oven at 85 ° C. for 4 to 5 hours, after which the dried cell walls were ground and ground in a coffee grinder prior to analysis. The ergosterol content of cell wall samples was then determined using sample preparation and extraction protocols, which are sourced from Dimartino (2007) and Huang et al (2009).

Table 1: Dry matter balance during fractionation of normal baker's yeast ( ^* : Lallemand's yeast culture collection)

表２：出発酵母（starting yeast）および酵母細胞壁画分中のエルゴステロール含量 The results of dry mass balance analysis during fractionation experiments with normal baker's yeast are shown in Table 1. A liquid cell wall of 2264 g was produced from 6000 g of liquid yeast 14.99% solid. Based on the solids content of the liquid cell wall, determined at 17.18%, the dry matter of the produced yeast cell wall amounts to 369 g. The yields achieved with cell wall and yeast extract were 43.25% and 56.75%, respectively. Both yields are similar to those achieved in the production of commercial yeast extracts by S. cerevisiae.

Table 2: Ergosterol content in starting yeast and yeast cell mural fractions

表３：分別中のエルゴステロール物質収支解析 As shown in Table 2, the ergosterol content of the starting yeast and the cell mural fraction produced was determined to be 0.704% and 1.603%, respectively. Thus, the ergosterol content of the cell wall was about 2.3 times higher than the ergosterol content of the starting yeast.

Table 3: Ergosterol mass balance analysis during fractionation

  The ergosterol mass balance analysis during the yeast fractionation experiment is shown in Table 3. The recovery efficiency of ergosterol in the cell wall was very close to 100%. The ergosterol recovered in the cell wall fraction accounted for 98.56% of the total ergosterol from the starting yeast, which was mainly divided by the ergosterol into the cell wall fraction during the yeast autolysis and fractionation process It is shown that. As a result, a dramatic enrichment of ergosterol in the cell wall fraction was achieved during the yeast extract production process.

Example 2: UV treatment of liquid yeast cell walls in a surface light bioreactor Liquid yeast cell walls (from Saccharomyces cerevisiae) were prepared similar to the protocol of Example 1. The dry matter content of the resulting liquid cell wall was adjusted to 11% prior to UV treatment.

  According to the process shown in FIG. 3, liquid yeast cell walls were UV treated using a surface ultraviolet light bioreactor as shown in FIG. At the heart of the photobioreactors facility is UVP's 8W UV lamp, which has three switchable UV tubes, short (254 nm), medium (302 nm) and long (365 nm) . Since the liquid yeast wall is nearly opaque to ultraviolet light, it was necessary to stir the yeast cell wall during processing so that all molecules of ergosterol reach the surface of the solution and are irradiated with ultraviolet light. A surface photobioreactor was used to achieve a thin layer of liquid yeast cell walls and increase the efficiency of vitamin D2 conversion. 120 mL of liquid yeast cell walls were placed in a rectangular container and irradiated continuously for 4 hours. During treatment, the liquid cell walls were mixed continuously. These experiments were performed at 4 ° C. and 50 ° C., and photochemical reactions were performed at a UV wavelength of 254 nm (UVC) for each temperature.

表４：２５４ｎｍでの表面光バイオリアクターにおけるＵＶ光処理後の、酵母細胞壁のビタミンＤ２含量 After treatment, dry cell wall samples were prepared. The liquid cell mural fraction was filtered or centrifuged and pressed to produce cell wall clumps. The cell wall clumps were then dried in an oven at 85 ° C. for 4 to 5 hours and further ground in a coffee breaker into fine particles before analysis. Dried yeast cell wall samples were prepared from Covance Laboratories for HPLC vitamin D2 analysis using the official method 982.29 (Official Methods of Analysis of AOAC INTERNATIONAL (2000) 17th Edition, AOAC INTERNATIONAL, Gaithersburg, MD, USA). Sent to Inc. (Madison, Wis.).

Table 4: Vitamin D2 content of yeast cell walls after UV light treatment in a surface light bioreactor at 254 nm

  As shown in Table 4, the vitamin D2 content in the control cell wall was judged to be 5570 IU / 100 g. This is because the commercial cream yeast used to produce the cell wall was obtained from plants containing about 3778 IU / 100 g vitamin D2 (on a dry matter basis). For vitamin D yeast cell wall samples produced at 4 ° C., the vitamin D2 content is 3,910,000 IU / 100 g (on a dry cell wall basis), which represents a 702-fold increase compared to the control cell wall. ing. At a temperature of 50 ° C., the vitamin D2 content is 5,800,000 IU / 100 g (on a dry cell wall basis), which represents a 1041 fold increase. Thus, the vitamin D2 content of liquid yeast cell walls can increase dramatically after UV treatment at 254 nm, especially at a temperature of 50 ° C.

表Ａ：水中光バイオリアクターにおいて液体細胞壁を４℃で２５４ｎｍのＵＶ処理を行う間のビタミンＤ２増加の経時変化 Example 3: UV treatment of liquid yeast cell walls in an underwater light bioreactor A liquid yeast cell wall (produced according to Example 1 from Saccharomyces cerevisiae) according to the process shown in FIG. The reactor was UV treated. A 20 liter light bioreactor equipped with a 14 watt ultraviolet lamp with a wavelength of 254 nm (UVC) of 18 liters of liquid yeast cell wall, the liquid cell wall being prepared according to example 1 and having a solid content of 10% Was put in Agitation was performed vigorously to ensure full UV treatment of the volume and to maintain high UV transmission by preventing the possibility of deposits around the quartz sleeve. Eighteen liters of liquid cell walls were continuously mixed and UV treated for 72 hours at 4 ° C.

Table A: Temporal change of vitamin D2 increase during UV treatment of 254 nm at 4 ° C in liquid cell wall in underwater light bioreactor

  As shown in Table A, the vitamin D2 content in the cell wall increases from 6590 IU / 100 g to 3,180,000 IU / 100 g in 72 hours, which represents an increase of about 485-fold. Although the vitamin D2 content achieved is lower than in the surface photobioreactors because of the large volume, aging shows that higher amounts of vitamin D2 were obtained over longer treatment times. Thus, the production of UV-treated yeast cell walls containing vitamin D2 by UV treatment of liquid yeast cell walls is compatible with both surface light bioreactors and underwater light bioreactors.

表５：表面光バイオリアクターにおけるＵＶ処理後の、酵母細胞壁中のビタミンＤ２の増加に対するＵＶ波長の影響 Example 4: Influence of UV wavelength on the increase of vitamin D2 in yeast cells A liquid yeast cell wall was produced according to Example 1 from Saccharomyces cerevisiae. UV treatment of the liquid yeast cell wall was performed using three different wavelengths: 254 nm (UVC), 302 nm (UVB), 365 nm (UVA) using the same surface light bioreactor as described in Example 2. . UV treatment was performed for 2 hours and all experiments were performed at room temperature. Four UV-treated dry yeast cell wall samples (three were treated, one as a control) were produced and sent to Covance for vitamin D2 analysis as described in Example 2.

Table 5: Influence of UV wavelength on the increase of vitamin D2 in yeast cell wall after UV treatment in surface light bioreactor

  As shown in Table 5, the selected UV wavelengths have a significant effect on the increase in vitamin D2 in yeast cell wall fractions. While UVA was not effective in converting ergosterol to vitamin D2 in liquid yeast cell walls, a wavelength of 302 nm (UVB) was found to be significantly more effective than 254 nm (UVC).

表６：表面光バイオリアクターにおける２５４ｎｍでのＵＶ処理済みトルラ酵母細胞壁のビタミンＤ２含量 Example 5 UV treatment of liquid yeast cell walls from non-Saccharomyces yeast in a surface light bioreactor UV treatment effectively increases the vitamin D 2 content of non-Saccharomyces yeast cell walls, UV treatment of liquid yeast cell walls made from Torula , According to the protocol described in Example 2. UV treatment was performed at 254 nm for 2 hours at two temperatures (4 ° C. and 50 ° C.).

Table 6: Vitamin D2 content of UV-treated Torula yeast cell walls at 254 nm in a surface light bioreactor

  Since Torula yeast cream was produced in the laboratory, vitamin D2 was not detected in the control Torula yeast cell wall. Similar to Saccharomyces cerevisiae yeast cell walls, vitamin D2 content of Torula yeast cell walls increases dramatically through UV treatment, and at 4 ° C from undetectable to 1,330,000 IU / 100 g (dry cell wall basis) Also at 50 ° C., from undetectable to an increase of up to 1,780,000 IU / 100 g (dry cell wall basis). In addition, at higher photochemical reaction temperatures (50 ° C.), higher vitamin D2 content was observed, consistent with what was previously observed in UV-treated yeast cell walls from Saccharomyces cerevisiae.

Example 6: Production of UV-treated yeast cell wall containing vitamin D2 through autolysis and fractionation of UV-treated yeast containing vitamin D2-pilot test UV-treated yeast containing vitamin D2 according to the process shown in Figure 4 Cell walls were produced by UV treatment of commercially produced liquid yeast to increase vitamin D2 content. The resulting UV-treated liquid yeast was then subjected to a typical yeast extract production process, which resulted in UV-treated yeast extract and UV-treated yeast cell wall (by-product). The treatment was carried out in a full scale underwater light bioreactor system as shown in FIG. The UV-treated liquid yeast containing vitamin D2 was further processed to a UV-treated concentrate instant dry yeast (IDY) (about 95% solids) containing vitamin D2 using a fluid bed dryer.

表７：ビタミンＤ２を含むＵＶ処理済み酵母のパイロット分別中における、ビタミンＤ２を含むＵＶ処理済み酵母抽出物およびビタミンＤ２を含むＵＶ処理済み酵母細胞壁画分の収率

表８：ビタミンＤ２を含むＵＶ処理済み酵母の分別後の吹き付け乾燥されたサンプルの物質収支 A pilot fractionation experiment of UV-treated yeast containing vitamin D2 was carried out with a UV-treated concentrate IDY containing vitamin D2 in a 75 liter Kemapach fermenter (75 L total volume). 10 kg of UV-treated concentrate IDY (vitamin D2 content: 2,700,000 IU / 100 g) containing vitamin D2 was blended with 50 kg of tap water to make cream yeast with a solids content of about 16%. 20 g of antifoam was added to the solution. Papain (Promod 144 GL from Biocatalysis) was added at a dose of 0.5% based on yeast dry matter. The yeast cream was hydrolyzed at 56 ° C. for 22 hours at a stirring speed of 600 revolutions per minute (rpm), pH 5.5 to 5.8. The resulting hydrolyzate was then inactivated by heat treatment at 80 ° C. for 60 minutes. After cooling, using a centrifuge to wash and collect the UV treated yeast cell wall containing vitamin D2 and the UV treated yeast containing vitamin D2 extract fractions. A separation has been performed. Then, final cell mural spray drying was performed to produce the final UV-treated dry yeast cell wall containing vitamin D2.

Table 7: UV-treated yeast extract containing vitamin D2 and UV-treated yeast cell mural fraction containing vitamin D2 during pilot fractionation of UV-treated yeast containing vitamin D2

Table 8: Mass balance of spray dried samples after fractionation of UV-treated yeast containing vitamin D2

  As shown in Table 7, UV-treated concentrate IDY containing vitamin D2, from 10 kg, UV-treated dry yeast cell wall containing vitamin D2 is 4.53 kg, and UV-treated dry yeast extract containing vitamin D2 is 4. 94 kg were produced. The yields of UV-treated yeast cell wall containing vitamin D2 and UV-treated yeast extract containing vitamin D2 were 47.84% and 52.76%, respectively.

  The vitamin D2 content of the UV-treated dry yeast cell wall and the UV-treated dry yeast extract was determined to be 5,360,000 IU / 100 g and 214,000 IU / 100 g, respectively. The vitamin D2 content in the UV-treated yeast cell fraction produced was 1.88 times higher than the vitamin D2 content in the UV-treated concentrate IDY. Table 8 also shows that during the UV-treated yeast extraction process from UV-treated concentrate IDY (90%) containing vitamin D2, vitamin D2 is mainly split into UV-treated yeast cell mural fractions This is in line with previous results that ergosterol was mainly divided into yeast cell mural fractions.

表９：ビタミンＤ２を含むＵＶ処理済み酵母の分別後の、吹き付け乾燥したサンプルの物質収支 Example 7: Production of UV-treated yeast cell wall with vitamin D2 through autolysis and fractionation of UV-treated yeast with vitamin D2-Commercial trial In a commercial trial, a 1500 liter fermentor was used. The first UV-treated liquid yeast containing vitamin D2 was produced according to Example 6. The solids and vitamin D2 content of the original UV-treated yeast solution was determined to be 17.84% and 2,560,000 IU / 100 g (dry yeast basis), respectively. 1300 liters of UV-treated liquid yeast containing vitamin D2 were hydrolyzed in the fermenter at 55 ° C. for 22 hours, stirring at 300 rpm, at a pH of 5.5. In addition to the mechanical defoamer, 500 mL of antifoam was also added. Papain (Promod 144 GL from Biocatalysis) was added at a dose of 0.5% based on yeast dry matter. After hydrolysis, the hydrolyzate was inactivated by heat treatment at 75 ° C. for 30 minutes. After cooling, separation was performed using a BTPX 205 separator (Alfa Laval Inc.) to collect yeast cell wall fractions.

Table 9: Mass balance of spray dried samples after fractionation of UV-treated yeast containing vitamin D2

  127 kg dry cell wall and 133 kg dry yeast extract from 1300 liters of UV-treated liquid yeast containing vitamin D2 (dry matter content 17.84%) or 260 kg UV-treated dry yeast (95% dry matter content) Was produced. The yields of yeast cell wall and yeast extract were 48.75% and 51.15%, respectively. The vitamin D2 content in the yeast cell wall was determined to be 4,980,000 IU / 100 g, which is the first vitamin D2 content in UV-treated liquid yeast containing vitamin D (2,560,000 IU / 100 g) It was 1.94 times higher. The vitamin D2 content in the yeast extract fraction was only 130,000 IU / 100 g. Thus, it is possible to produce yeast cell walls containing commercial levels of vitamin D2 with potentially higher vitamin D2 content than found in yeast containing vitamin D2.

Example 8: Effect of supplementing UV-treated yeast cell wall containing vitamin D2 with the diet of laying hens on egg yolk's vitamin D2 content 126 39-week-old egg laying chickens (shavers) The university (Montreal, Canada) was randomly assigned to seven diets over a 10 week experimental period. All chickens were caged (6 cage replicates per therapy; n = 3 chickens / cage), lighted for 15 hours a day at a temperature of 22.0 ° C ( lightning) were grown under controlled conditions. The following seven diets were examined: diet 1 = diet without vitamin D (-CTL, negative control); diet 2 = diet 1 + vitamin D inactivated yeast (2,500 IU / kg); diet 3 = diet 1 + vitamin D. Inactivated yeast (15,000 IU / kg); Diet 4 = Diet 1 + UV treated yeast cell wall with vitamin D (2,500 IU / kg); Diet 5 = Diet 1 + UV treated yeast cell wall with vitamin D (15 , 000 IU / kg).

  All chickens were fed a vitamin-free diet supplemented with 2,500 kg IU / kg vitamin D3 prior to treatment. After a two week adaptation period, samples were collected. Feed and water were provided ad libitum.

  Eggs were collected and weighed daily. Chicken weight and feed intake were measured in cages at two week time intervals. Two eggs per cage (n = 12 / treatment) were collected every two weeks to measure eggshell strength. Another set of eggs was collected simultaneously to assess the vitamin D content of egg yolk. Blood samples were also collected for serum vitamin D analysis (n = 6 / treatment). The vitamin D3 and D2 content in egg yolk samples was analyzed by Heartland Assays, located in Ms. Iowa. Serum samples were sent to Intelimmune LLC Inc., West Lafayette, IN for 25- (OH) D2 and 25- (OH) D3 analysis using LC / MS / MRM.

  At the end of the study, 10 chickens per diet were bled and euthanized for the carotid artery, and the tibia of each chicken was collected for measurement of tibia length, diameter and breaking strength. The The fracture strength of the eggshell and tibia was measured as a compressive fracture force using a tensiometer (Instron Model 4500, MA, USA) fitted with a 500-N load cell.

表Ｂ：産卵鶏のビタミンＤを補充したさまざまな食事が卵黄のビタミンＤ２含量に与える影響 Based on data collected for body weight, feed intake, feed efficiency, egg production rate, egg weight, egg diameter, and eggshell breakage strength during the study (data not shown) No significant effects were observed in the 5 diets.

Table B: Effects of various diets supplemented with vitamin D on laying hens on vitamin D2 content of egg yolk

  The highest vitamin D2 content in egg yolk is achieved with a diet containing UV-treated yeast cell walls containing 15,000 IU / kg of vitamin D2 (diet 5), the vitamin D2 content in egg yolk after 6 weeks, It was 422 IU / 100 g. The total egg-based vitamin D2 level was 146 IU / 100 g (see Factor Yolk Divided by Factor 2.9: Mattila et al., 2004). On an egg basis (assuming an egg weight of 60 g), the maximum vitamin D2 content achieved per egg is about 87.6 IU per egg, this means that one person can have eggs daily It represents 175.2 IU vitamin D2 intake (or 44.4% RDA) per day when two are eaten. In this study it was well documented that yeast cell walls containing vitamin D2 can be used to supplement the diet of laying hens to increase the vitamin D2 content of eggs.

  Although the present invention has been described in relation to particular embodiments thereof, it is understood that further modifications are possible, and generally in accordance with the principles of the present invention, known habits or conventions within the scope of the art to which the present invention belongs. It is intended to cover any variations, uses, or adaptations of the present invention as may be applied to the essential features described above, including deviations from the present disclosure, and according to the claims. It will be understood that the present application is intended.

[Aspect of embodiment]
(1) Yeast cell wall containing vitamin D2.
(2) In the yeast cell wall according to embodiment 1,
The yeast cell wall, wherein said yeast is from Saccharomyces or is any non-Saccharomyces yeast.
(3) In the yeast cell wall according to embodiment 2,
The non-saccharomyces yeast is a yeast cell wall selected from the group consisting of Candida species, Hansenia spora species, Hansenula species, Clyberomyces species, methynikobia species, Pichia species, Starmerella species, and Truluspora species.
(4) In the yeast cell wall according to embodiment 3,
The yeast cell wall, wherein the yeast is Saccharomyces cerevisiae, or Cyberlindnera jadinii (Torula yeast).
(5) In the yeast cell wall according to any one of the embodiments 1 to 4,
The vitamin D2 is added vitamin D2, yeast cell wall.

(6) In the yeast cell wall according to any one of the embodiments 1 to 4,
The yeast cell wall is UV treated, yeast cell wall.
(7) In the UV-treated yeast cell wall according to embodiment 6,
The UV-treated yeast cell wall comprises a β-glucan-containing UV-treated yeast cell wall.
(8) In the UV-treated yeast cell wall according to embodiment 7,
UV-treated yeast wherein the content of said β-glucan is at least 75%, preferably 80%, more preferably 85%, most preferably 90% of the content of β-glucan in the yeast cell wall not treated with UV Cell wall.
(9) Use of a yeast cell wall according to any of embodiments 1-8 as a dietary supplement for nutrition of animals or humans.
(10) Use of the yeast cell wall according to any one of the embodiments 1 to 8 as an additive for baked goods, functional foods, diet foods, nutritional foods, fermented beverages or non-fermented beverages.

(11) A baked confectionery produced using the yeast cell wall according to any one of embodiments 1 to 8.
(12) In the baked good according to the embodiment 11,
The baked confectionery is a bread, a cracker, a stick for sports, or a biscuit.
(13) In the composition,
Animal feed,
9. A yeast cell wall according to any of embodiments 1-8,
Composition containing.
(14) In the composition according to embodiment 13,
A composition for use in the diet of laying hens to increase vitamin D2 content in egg yolk.
(15) In the composition according to embodiment 13,
A composition for use in the diet of animals as a dietary supplement.

(16) In a method of increasing vitamin D2 content in egg yolk,
Providing the laying hens with a composition comprising a diet of the laying hens and a UV-treated yeast cell wall comprising vitamin D2.
(17) A method of preparing UV-treated yeast cell wall containing vitamin D2,
Supplying yeast;
Autodigesting and / or hydrolyzing said yeast to obtain autodigestion and / or hydrolysis yeast;
Separating the autolyzed and / or hydrolyzed yeast to provide a yeast cell wall fraction and a yeast extract;
Treating the yeast cell mural fraction with UV light to obtain a UV-treated yeast cell wall comprising the vitamin D2;
Method, including.
(18) In a method of preparing UV-treated yeast cell wall containing vitamin D2,
Supplying yeast;
Treating the yeast with UV light to obtain a UV-treated yeast comprising vitamin D2.
Autolyzing and / or hydrolyzing the UV-treated yeast containing vitamin D2 to obtain an auto-digested and / or hydrolyzed UV-treated yeast containing vitamin D2;
Separating the autolyzed and / or hydrolyzed UV-treated yeast containing vitamin D2 to provide a UV-treated yeast cell mural fraction containing yeast and a yeast extract;
Method, including.
(19) In the method according to embodiment 18,
The method wherein the content of the vitamin D2 in the UV-treated yeast cell wall is at least 50 times higher, preferably at least 5000 times, or most preferably at least 10000 times higher than the non-UV-treated yeast cell wall.
(20) In the method according to embodiment 18,
The content of the vitamin D2 in the UV-treated yeast cell wall is at least 1.25 times, preferably at least 1.5 times, more preferably at least 1.5 times the content of the vitamin D2 in the UV-treated yeast containing the vitamin D2. 1.75 times, most preferably at least 2 times the method.

(21) In the method according to any one of embodiments 18 to 20,
The method wherein the yeast is from Saccharomyces or is any non-Saccharomyces yeast.
(22) In the method according to embodiment 21,
The method, wherein the non-saccharomyces yeast is selected from the group consisting of Candida species, Hansenia spora species, Hansenula species, Clyberomyces species, methynikobia species, Pichia species, Starmerella species, and Torluspora species.
(23) In the method according to embodiment 18,
The method, wherein the yeast is Saccharomyces cerevisiae, or Cyberlindnera jadinii (Torula yeast).

Fig. 6 shows a surface UV light bioreactor used for UV treatment of yeast cell walls according to an embodiment of the present invention. Fig. 16 shows a UV light bioreactor in water for UV treatment of yeast cell walls with volumes of up to 20 L according to another embodiment of the present invention. FIG. 6 illustrates a method of producing a UV-treated yeast cell wall comprising vitamin D2 according to an embodiment of the present invention. Fig. 6 shows an alternative method of producing UV-treated yeast cell wall comprising vitamin D2 according to another embodiment of the present invention. FIG. 7 shows β-glucan content in yeast cell walls before and after treatment with UV light according to an embodiment of the present invention.

Claims (1)

  Yeast cell wall containing vitamin D2.

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