TWI839198B - Method for producing vitamin d content - Google Patents

Abstract

The present invention discloses a method for producing vitamin D content comprising illuminating a probiotic bacterium by using a illuminative light source for a first time so as to obtain vitamin D, wherein the probiotic bacterium comprises a yeast, a lactic acid bacterium or an acetic acid bacterium, and the illuminative light source comprises natural light or ultraviolet light. The method disclosed herein promotes the probiotic bacterium to produce vitamin D through illuminating the probiotic bacterium. During the process, bacteria culture is not required, which simplifies manufacturing procedure of vitamin D, and improving steady production as well as food safety of probiotic product.

Description

Method of Producing Vitamin D Content

The present invention relates to a method for producing vitamins, and in particular to the production of vitamin D, and a production method using microorganisms as a production platform.

As we all know, vitamin D is a fat-soluble vitamin that helps the body absorb calcium, which is very beneficial for maintaining bone health and preventing osteoporosis or fractures. In addition, vitamin D also has the effects of enhancing the immune system function and reducing inflammatory responses, such as enhancing adjuvant cells, inhibiting Th1 cell activity or promoting Th2 cell activity. It is also involved in promoting cell growth, cell differentiation, reducing the risk of heart disease, and inhibiting the mechanism of carcinogenesis.

Vitamin D is mainly taken from food or synthesized by the human body. For example, vitamin D2 can be taken from food or vitamin D supplements, while vitamin D3 can be converted and produced in skin cells under sunlight. Natural sources of vitamin D mainly include animal sources such as salmon, tuna, sardines or oysters, and non-animal sources such as edible fungi such as shiitake mushrooms, mushrooms, and button mushrooms. The sources of intake are quite limited.

In recent years, due to environmental issues and the rise of the trend of dietary health, the number of vegetarians has increased day by day. In the absence of animal sources of vitamin D, edible or medicinal fungi have become an important source of vitamin D in natural foods, but the content cannot meet the human body's vitamin D needs. In addition, with the changes in modern lifestyles, people's exposure to sunlight is becoming shorter and shorter, and the popularization of sun protection concepts has resulted in the skin's exposure to sunlight still being far from enough to synthesize sufficient vitamin D, all of which are potential threats to people's health.

The known industrial production of synthetic vitamins is to add raw materials such as fungi or yeast to special culture media for metabolic reactions, and use fermentation reactions and photocatalysis to convert the precursors (such as ergosterol) in these raw materials into vitamin D, and use the synthesized vitamin D as an additive or supplement. These methods involve the adjustment of culture medium ingredients and fermentation conditions, which increases the complexity of the production process and uncontrollable risks. Not only is it difficult to control the output, but it is also difficult to ensure the food safety of the end product.

The purpose of the present invention is to provide a method for producing vitamin D content, which has a simple process and is easy to operate. It can be widely used in the production of products added with vitamin D in the food industry to increase the vitamin D intake of consumers, so as to achieve specific effects such as preventing osteoporosis, immune regulation, and cancer prevention. The method includes an irradiation treatment, which includes irradiating a probiotic with an irradiation light source for a first time to obtain vitamin D, wherein the irradiation light source includes natural light or ultraviolet light, and the probiotic includes a yeast, a lactic acid bacterium, or an acetic acid bacterium.

The method as described above, wherein the yeast comprises Saccharomyces cerevisiae , Kluyveromyces lactis , Kluyveromyces marxianus , Pichia membranifaciens , Pichia kudriavzevii , Lanchancea fermentati , Lanchancea meyercii , Kazachstania aerobia or Kazachstania unispora or any combination of two or more thereof.

The method as described above, wherein the lactic acid bacteria include Lactobacillus kefiri , Lactobacillus kefiranofaciens , Lactobacillus helveticus , Lactococcus lactis , Lactobacillus brevis , Lactobacillus buchneri , Lactobacillus casei subsp . casei , Lactobacillus casei subsp . rhamnosus , Lactobacillus fermentum , Lactobacillus mali , Lactobacillus nagelli , Lactobacillus paracasei, Lactobacillus spp. Lactobacillus paracasei ), Lactobacillus plantarum , Lactobacillus acidophilus , or any combination of two or more thereof.

The method as described above, wherein the acetic acid bacteria comprises Acetobacter fabarum , Acetobacter orientalis , Acetobacter lovaniensis , Acetobacter aceti , Acetobacter rasens or any combination of two or more thereof.

Another object of the present invention is to provide a method for producing vitamin D, which includes an irradiation treatment, which includes irradiating a probiotic with an irradiation light source for a first time to obtain vitamin D, wherein the probiotic includes a yeast and a lactic acid bacterium.

The method as described above, wherein the yeast comprises Saccharomyces cerevisiae , Kluyveromyces lactis , Kluyveromyces marxianus , Pichia membranifaciens , Pichia kudriavzevii , Lanchancea fermentati , Lanchancea meyercii , Kazachstania aerobia or Kazachstania unispora , and the lactic acid bacteria comprises Lactobacillus kefiri , Lactobacillus kefiranofaciens , Lactobacillus helveticus ), Lactococcus lactis, Lactobacillus brevis , Lactobacillus buchneri , Lactobacillus casei subsp . casei , Lactobacillus casei subsp . rhamnosus , Lactobacillus fermentum , Lactobacillus mali , Lactobacillus nagelli , Lactobacillus paracasei , Lactobacillus plantarum , Lactobacillus acidophilus , or any combination of two or more thereof.

The method as described above, wherein the probiotic further comprises an acetic acid bacterium, which includes Acetobacter fabarum , Acetobacter orientalis , Acetobacter lovaniensis , Acetobacter aceti or Acetobacter rasens .

In various embodiments of the present invention, before the irradiation treatment, the method further includes a temperature control treatment: placing the probiotics at a temperature-controlled temperature for a second time, the temperature-controlled temperature is less than or equal to the human body's basal temperature but not zero degrees Celsius, and the second time is greater than or equal to the first time.

In various embodiments of the present invention, the irradiation treatment further comprises: before the irradiation light source irradiates the probiotics, placing the probiotics at a distance from the irradiation light source, wherein the irradiation distance is 2 to 10 centimeters.

In various embodiments of the present invention, the temperature control temperature includes a first temperature or a second temperature, the first temperature is less than or equal to the basal temperature of the human body, and the second temperature is less than the first temperature but not zero degrees Celsius.

In various embodiments of the present invention, the irradiation intensity of the UV light source is 0.2 to 1.0 mW/cm ² .

In various embodiments of the present invention, the first time is at least 30 minutes, and the second time is 60 to 120 minutes.

The method for producing vitamin D content provided by the present invention promotes the conversion of probiotics into vitamin D by irradiating them with light, and increases the yield of vitamin D by treating the probiotics with temperature control. The process does not involve any front-end probiotic cultivation, thereby reducing the complexity of the production process and improving the safety and reliability of the terminal product.

In order to fully understand the present invention, preferred embodiments are given below and described in detail with reference to the accompanying drawings, which are not intended to limit the implementation of the present invention.

Please refer to FIG. 1A , which illustrates the process steps of a first embodiment of a method for producing vitamin D content provided by the present invention, the method comprising an irradiation treatment, which comprises irradiating a probiotic with an irradiation light source for a first time to obtain vitamin D, wherein the probiotic comprises a yeast, a lactic acid bacterium or a combination thereof, and the irradiation light source comprises natural light or ultraviolet light; Specifically, the natural light can be ambient light, sunlight, etc., wherein the ambient light is not particularly limited and can be provided by a common light source such as a fluorescent lamp, an incandescent lamp, a light emitting diode or a halogen lamp. The ultraviolet light is light with a wavelength between 10 and 400 nanometers, and the light source can be, for example, a black light, a fluorescent lamp or a mercury lamp. In many embodiments, the ultraviolet light includes UVA, UVB or UVC.

Specifically, the irradiation treatment is to place the probiotics under a light source for continuous irradiation to stimulate the conversion and synthesis mechanism of vitamin D precursors in the probiotics, such as stimulating ergosterol, to further promote the production of vitamin D. According to tests, the vitamin D content of the probiotics after irradiation treatment can be expected to reach 10 to 30 μg/100 μg. The length of irradiation time affects the production of vitamin D. In these embodiments, the first time is at least 30 minutes, preferably 60 to 70 minutes.

In these embodiments, the probiotics can be a single bacterial consortium or a composite bacterial consortium, and their appearance is not particularly limited, for example, they can be dried bacterial blocks, dried bacterial powder or bacterial liquid, wherein the drying method of the bacterial blocks or bacterial powder is not particularly limited, for example, they can be washed and then drained, washed and then dried, washed and then air-dried, or washed and then dried in the shade.

Specifically, the yeast may be Saccharomyces cerevisiae , Kluyveromyces lactis , Kluyveromyces marxianus , Pichia membranifaciens , Pichia kudriavzevii , Lanchancea fermentati , Lanchancea meyercii , Kazachstania aerobia or Kazachstania unispora ; the lactic acid bacteria may be Lactobacillus kefiri , Lactobacillus kefiranofaciens , Lactobacillus helveticus. Lactobacillus helveticus 、 Lactococcus lactis 、 Lactobacillus brevis 、 Lactobacillus buchneri 、 Lactobacillus casei subsp . casei 、 Lactobacillus casei subsp . rhamnosus 、 Lactobacillus fermentum 、 Lactobacillus mali 、 Lactobacillus nagelli 、 Lactobacillus paracasei 、 Lactobacillus plantarum 、 Lactobacillus acidophilus ); the added yeast and lactic acid bacteria have probiotic effects. In this embodiment, they can provide a rich content of vitamin D in addition to the effect of regulating the intestines and strengthening the stomach in the terminal product.

In some embodiments, the light source is ultraviolet light with a wavelength between 100 and 400 nanometers, specifically ultraviolet light A (UVA), ultraviolet light B (UVB) or ultraviolet light C (UVC), and the specific wavelength of the ultraviolet light irradiated each time is not limited. It is well known in the art that UVA can be classified into UVA-1 between 340 and 400 nanometers and UVA-2 between 320 and 340 nanometers according to the wavelength, the wavelength of UVB is between 280 and 320 nanometers, and the wavelength of UVC is between 100 and 280 nanometers. Among them, ultraviolet light with a wavelength below 200 nanometers is easily absorbed by the air. Therefore, in these embodiments, the wavelength of ultraviolet light is preferably 200 to 400 nanometers, and more preferably 250 to 340 nanometers.

In these embodiments, different irradiation intensities are required under light sources with different wavelength properties. The preferred irradiation intensity is 0.2 to 1.0 mW/cm ² , and more preferably 0.4 to 0.6 mW/cm ² . It can be understood that the probiotics are stationary under the irradiation of the light source and gradually convert and synthesize vitamin D. The total exposure in the first time is a key factor affecting the final vitamin D yield. Specifically, the total exposure is at least 24,000 KJ/cm ² , and preferably 14,400 to 25,200 KJ/cm ² .

Furthermore, under the aforementioned total exposure, the total energy that ultimately reaches the probiotics is affected by the irradiation distance. Generally speaking, the closer the distance between the irradiation light source and the irradiated object is, the more total energy the irradiated object captures. However, if the distance between the irradiated object and the irradiation light source is too close, the precursors in the irradiated object may be destroyed by light due to excessive energy capture, thereby reducing the yield of the final product. Therefore, in these embodiments, the irradiation distance is preferably 2 to 10 cm, and more preferably 3 to 5 cm.

In other embodiments, the probiotics are defined by a specific combination of two or more species, wherein, for example, a combination of a single yeast species and a single lactic acid bacteria species, the probiotics include brewing yeast ( Saccharomyces cerevisiae ) and Lactobacillus kefiri , brewing yeast ( Saccharomyces cerevisiae ) and Lactobacillus kefiranofaciens , brewing yeast ( Saccharomyces cerevisiae ) and Lactobacillus helveticus , brewing yeast ( Saccharomyces cerevisiae ) and Lactococcus lactis , brewing yeast ( Saccharomyces cerevisiae ) and Lactobacillus brevis ), Saccharomyces cerevisiae and Lactobacillus buchneri , Saccharomyces cerevisiae and Lactobacillus casei subsp. Casei , Saccharomyces cerevisiae and Lactobacillus casei subsp. Rhamnosus , Saccharomyces cerevisiae and Lactobacillus fermentum , Saccharomyces cerevisiae and Lactobacillus mali , Saccharomyces cerevisiae and Lactobacillus nagelli ), Saccharomyces cerevisiae and Lactobacillus paracasei , Saccharomyces cerevisiae and Lactobacillus plantarum , Saccharomyces cerevisiae and Lactobacillus acidophilus , Kluyveromyces lactis and Lactobacillus kefiri , Kluyveromyces lactis and Lactobacillus kefiranofaciens , Kluyveromyces lactis and Lactobacillus helveticus , Kluyveromyces Kluyveromyces lactis and lactococcus lactis , Kluyveromyces lactis and Lactobacillus brevis , Kluyveromyces lactis and Lactobacillus buchneri , Kluyveromyces lactis and Lactobacillus casei subsp. Casei , Kluyveromyces lactis and Lactobacillus casei subsp. Rhamnosus , Kluyveromyces lactis and Lactobacillus fermentum , Kluyveromyces lactis ) and Lactobacillus mali , Kluyveromyces lactis and Lactobacillus nagelli , Kluyveromyces lactis and Lactobacillus paracasei , Kluyveromyces lactis and Lactobacillus plantarum , Kluyveromyces lactis and Lactobacillus acidophilus , Kluyveromyces marxianus and Lactobacillus kefiri , Kluyveromyces marxianus and Lactobacillus kefiri ) and Lactobacillus kefiranofaciens , Kluyveromyces marxianus and Lactobacillus helveticus , Kluyveromyces marxianus and Lactococcus lactis , Kluyveromyces marxianus and Lactobacillus brevis , Kluyveromyces marxianus and Lactobacillus buchneri , Kluyveromyces marxianus and Lactobacillus casei subsp. Casei , Kluyveromyces marxianus and Lactobacillus casei subsp. Casei Kluyveromyces marxianus and Lactobacillus casei subsp. Rhamnosus , Kluyveromyces marxianus and Lactobacillus fermentum , Kluyveromyces marxianus and Lactobacillus mali , Kluyveromyces marxianus and Lactobacillus nagelli , Kluyveromyces marxianus and Lactobacillus paracasei , Kluyveromyces marxianus and Lactobacillus plantarum ), Kluyveromyces marxianus and Lactobacillus acidophilus , Pichia membranifaciens and Lactobacillus kefiri , Pichia membranifaciens and Lactobacillus kefiranofaciens , Pichia membranifaciens and Lactobacillus helveticus , Pichia membranifaciens and lactococcus lactis , Pichia membranifaciens and Lactobacillus brevis , Pichia membranifaciens ) and Lactobacillus buchneri , Pichia membranifaciens and Lactobacillus casei subsp. Casei , Pichia membranifaciens and Lactobacillus casei subsp. Rhamnosus , Pichia membranifaciens and Lactobacillus fermentum , Pichia membranifaciens and Lactobacillus mali , Pichia membranifaciens and Lactobacillus nagelli , Pichia membranifaciens and Lactobacillus paracasei ), Pichia membranifaciens and Lactobacillus plantarum , Pichia membranifaciens and Lactobacillus acidophilus , Pichia kudriavzevii and Lactobacillus kefiri , Pichia kudriavzevii and Lactobacillus kefiranofaciens , Pichia kudriavzevii and Lactobacillus helveticus , Pichia kudriavzevii and lactococcus lactis , Pichia kudriavzevii and Lactobacillus brevis , Pichia kudriavzevii and Lactobacillus buchneri , Pichia kudriavzevii and Lactobacillus casei subsp. Casei , Pichia kudriavzevii and Lactobacillus casei subsp. Rhamnosus , Pichia kudriavzevii and Lactobacillus fermentum , Pichia kudriavzevii and Lactobacillus mali , Pichia kudriavzevii and Lactobacillus nagelli , Pichia kudriavzevii and Lactobacillus paracasei , Pichia kudriavzevii and Lactobacillus plantarum , Pichia kudriavzevii and Lactobacillus acidophilus , Lanchancea fermentati and Lactobacillus kefiri , Lanchancea fermentati and Lactobacillus kefiranofaciens , Lanchancea fermentati ) and Lactobacillus helveticus , Lanchancea fermentati and Lactococcus lactis , Lanchancea fermentati and Lactobacillus brevis , Lanchancea fermentati and Lactobacillus buchneri , Lanchancea fermentati and Lactobacillus casei subsp. Casei , Lanchancea fermentati and Lactobacillus casei subsp. Rhamnosus , Lanchancea fermentati and Lactobacillus fermentum , Lanchancea fermentati and Lactobacillus fermentum fermentati and Lactobacillus mali , Lanchancea fermentati and Lactobacillus nagelli , Lanchancea fermentati and Lactobacillus paracasei , Lanchancea fermentati and Lactobacillus plantarum , Lanchancea fermentati and Lactobacillus acidophilus , Lanchancea meyercii and Lactobacillus kefiri , Lanchancea meyercii and Lactobacillus kefiranofaciens ), Lanchancea meyercii and Lactobacillus helveticus , Lanchancea meyercii and Lactococcus lactis , Lanchancea meyercii and Lactobacillus brevis , Lanchancea meyercii and Lactobacillus buchneri , Lanchancea meyercii and Lactobacillus casei subsp. Casei , Lanchancea meyercii and Lactobacillus casei subsp. Rhamnosus , Lanchancea meyercii ) and Lactobacillus fermentum , Lanchancea meyercii and Lactobacillus mali , Lanchancea meyercii and Lactobacillus nagelli, Lanchancea meyercii and Lactobacillus paracasei , Lanchancea meyercii and Lactobacillus plantarum , Lanchancea meyercii and Lactobacillus acidophilus , aerobic Kazachstania aerobia and Lactobacillus kefiri ), aerobic Kazachstania aerobia and Lactobacillus kefiranofaciens , aerobic Kazachstania aerobia and Lactobacillus helveticus , aerobic Kazachstania aerobia and Lactococcus lactis , aerobic Kazachstania aerobia and Lactobacillus brevis , aerobic Kazachstania aerobia and Lactobacillus buchneri , aerobic Kazachstania aerobia and Lactobacillus casei subsp. Casei ), aerobic Kazachstania aerobia and Lactobacillus casei subsp. Rhamnosus , aerobic Kazachstania aerobia and Lactobacillus fermentum , aerobic Kazachstania aerobia and Lactobacillus mali , aerobic Kazachstania aerobia and Lactobacillus nagelli , aerobic Kazachstania aerobia and Lactobacillus paracasei , aerobic Kazachstania aerobia and Lactobacillus plantarum ), Kazachstania aerobia and Lactobacillus acidophilus , Kazachstania unispora and Lactobacillus kefiri , Kazachstania unispora and Lactobacillus kefiranofaciens , Kazachstania unispora and Lactobacillus helveticus , Kazachstania unispora and Lactococcus lactis , Kazachstania unispora and Lactobacillus brevis ), Kazachstania unispora and Lactobacillus buchneri, Kazachstania unispora and Lactobacillus casei subsp. Casei , Kazachstania unispora and Lactobacillus casei subsp . Rhamnosus , Kazachstania unispora and Lactobacillus fermentum , Kazachstania unispora and Lactobacillus mali , Kazachstania unispora and Lactobacillus longifolia Lactobacillus nagelli ), Kazachstania unispora and Lactobacillus paracasei , Kazachstania unispora and Lactobacillus plantarum , or Kazachstania unispora and Lactobacillus acidophilus .

In some other embodiments, the probiotic may also be a combination of a single yeast species and any two lactic acid bacteria species. For example, if the probiotic comprises Kluyveromyces marxianus , the probiotic species combination may include Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus kefiranofaciens , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus helveticus , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus helveticus. ) and Lactococcus lactis , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus brevis , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus buchneri, Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus casei subsp. casei , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus casei subsp. casei kefiri and Lactobacillus casei subsp. rhamnosus , Kluyveromyces marxianus, Lactobacillus kefiri and Lactobacillus fermentum , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus mali , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus nagelli , Kluyveromyces marxianus ), Lactobacillus kefiri and Lactobacillus paracasei , Kluyveromyces marxianus, Lactobacillus kefiri and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus kefiri and Lactobacillus acidophilus, Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus helveticus , Kluyveromyces marxianus Kluyveromyces marxianus ), Lactobacillus kefiranofaciens and Lactococcus lactis , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus brevis , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus buchneri , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus casei Lactobacillus casei subsp. casei ), Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus casei subsp. rhamnosus , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus fermentum , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus mali , Kluyveromyces marxianus ), Lactobacillus kefiranofaciens and Lactobacillus nagelli , Kluyveromyces marxianus, Lactobacillus kefiranofaciens and Lactobacillus paracasei , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Lactobacillus acidophilus. acidophilus , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactococcus lactis , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus brevis , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus buchneri , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus casei subsp. casei ), Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus casei subsp. rhamnosus , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus fermentum , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus mali , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus casei subsp. rhamnosus Lactobacillus nagelli , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus paracasei , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus helveticus , and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactococcus lactis , and Lactobacillus breve brevis ), Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus buchneri , Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus casei subsp. casei , Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus casei subsp . rhamnosus , Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus fermentum ), Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus mali , Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus nagelli , Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus paracasei , Kluyveromyces marxianus , Lactococcus lactis and Lactobacillus plantarum , Kluyveromyces marxianus Kluyveromyces marxianus ), Lactococcus lactis , and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus brevis , and Lactobacillus buchneri , Kluyveromyces marxianus , Lactobacillus brevis , and Lactobacillus casei subsp. casei , Kluyveromyces marxianus , Lactobacillus brevis , and Lactobacillus casei subsp. rhamnosus ), Kluyveromyces marxianus , Lactobacillus brevis and Lactobacillus fermentum , Kluyveromyces marxianus , Lactobacillus brevis and Lactobacillus mali , Kluyveromyces marxianus , Lactobacillus brevis and Lactobacillus nagelli , Kluyveromyces marxianus , Lactobacillus brevis and Lactobacillus paracasei ), Kluyveromyces marxianus , Lactobacillus brevis , and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus brevis , and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus buchneri , and Lactobacillus casei subsp. casei , Kluyveromyces marxianus , Lactobacillus buchneri , and Lactobacillus rhamnosus casei subsp. rhamnosus ), Kluyveromyces marxianus , Lactobacillus buchneri and Lactobacillus fermentum , Kluyveromyces marxianus , Lactobacillus buchneri and Lactobacillus mali , Kluyveromyces marxianus , Lactobacillus buchneri and Lactobacillus nagelli , Kluyveromyces marxianus , Lactobacillus buchneri and Lactobacillus nagelli ) and Lactobacillus paracasei , Kluyveromyces marxianus , Lactobacillus buchneri and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus buchneri and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus casei subsp. casei and Lactobacillus casei subsp. rhamnosus , Kluyveromyces marxianus ), Lactobacillus casei subsp. casei and Lactobacillus fermentum , Kluyveromyces marxianus , Lactobacillus casei subsp. casei and Lactobacillus mali , Kluyveromyces marxianus , Lactobacillus casei subsp. casei and Lactobacillus nagelli , Kluyveromyces marxianus , Lactobacillus casei subsp. casei and Lactobacillus paracasei ), Kluyveromyces marxianus , Lactobacillus casei subsp. casei and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus casei subsp . casei and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus casei subsp. rhamnosus and Lactobacillus fermentum , Kluyveromyces marxianus , Lactobacillus casei subsp. rhamnosus ) and Lactobacillus mali , Kluyveromyces marxianus , Lactobacillus casei subsp. rhamnosus and Lactobacillus nagelli , Kluyveromyces marxianus , Lactobacillus casei subsp. rhamnosus and Lactobacillus paracasei, Kluyveromyces marxianus , Lactobacillus casei subsp. rhamnosus and Lactobacillus plantarum, Kluyveromyces marxianus , Lactobacillus casei subsp. rhamnosus and Lactobacillus plantarum , marxianus ), Lactobacillus casei subsp. rhamnosus , and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus fermentum , and Lactobacillus mali , Kluyveromyces marxianus , Lactobacillus fermentum , and Lactobacillus nagelli , Kluyveromyces marxianus , Lactobacillus fermentum , and Lactobacillus paracasei , Kluyveromyces marxianus Kluyveromyces marxianus ), Lactobacillus fermentum and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus fermentum and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus mali and Lactobacillus nagelli , Kluyveromyces marxianus , Lactobacillus mali and Lactobacillus paracasei , Kluyveromyces marxianus Kluyveromyces marxianus ), Lactobacillus mali , and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus mali , and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus nagelli , and Lactobacillus paracasei , Kluyveromyces marxianus , Lactobacillus nagelli , and Lactobacillus plantarum , Kluyveromyces marxianus Kluyveromyces marxianus ), Lactobacillus nagelli , and Lactobacillus acidophilus , Kluyveromyces marxianus , Lactobacillus paracasei , and Lactobacillus plantarum , Kluyveromyces marxianus , Lactobacillus paracasei , and Lactobacillus acidophilus , or Kluyveromyces marxianus , Lactobacillus plantarum , and Lactobacillus acidophilus .

In some other embodiments, the probiotic comprises an acetic acid bacterium, which can be exemplified by Acetobacter fabarum , Acetobacter orientalis , Acetobacter lovaniensis , Acetobacter aceti or Acetobacter rasens .

In these embodiments, the probiotics further include a combination of individual single species of yeast, lactic acid bacteria and acetic acid bacteria. Specifically, the combination is kefir bacteria, which is a composite bacterial consortium of yeast, lactic acid bacteria and acetic acid bacteria. For example, kefir bacteria including brewing yeast ( Saccharomyces cerevisiae ) can be listed as brewing yeast ( Saccharomyces cerevisiae ), kefir Lactobacillus ( Lactobacillus kefiri ) and broad bean acetic acid bacteria ( Acetobacter fabarum ), brewing yeast ( Saccharomyces cerevisiae ), mare milk-like Lactobacillus ( Lactobacillus kefiranofaciens ) and broad bean acetic acid bacteria ( Acetobacter fabarum ), brewing yeast ( Saccharomyces cerevisiae ) ), Lactobacillus helveticus and Acetobacter fabarum, Saccharomyces cerevisiae, Lactococcus lactis and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus brevis and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus buchneri and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus casei subsp. Casei ) and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus casei subsp. Rhamnosus and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus fermentum and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus mali and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus nagelli ) and Acetobacter fabarum , Saccharomyces cerevisiae, Lactobacillus paracasei and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus plantarum and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus acidophilus and Acetobacter fabarum , Saccharomyces cerevisiae , Lactobacillus kefiri and Acetobacter orientalis ), brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus kefiranofaciens and Acetobacter orientalis, brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus helveticus and Acetobacter orientalis, brewing yeast ( Saccharomyces cerevisiae ), Lactococcus lactis and Acetobacter orientalis, brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus brevis and Acetobacter orientalis , brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus buchneri and Acetobacter orientalis , Saccharomyces cerevisiae , Lactobacillus casei subsp. Casei and Acetobacter orientalis , Saccharomyces cerevisiae, Lactobacillus casei subsp. Rhamnosus and Acetobacter orientalis , Saccharomyces cerevisiae , Lactobacillus fermentum and Acetobacter orientalis , Saccharomyces cerevisiae ), Lactobacillus mali and Acetobacter orientalis , Saccharomyces cerevisiae , Lactobacillus nagelli and Acetobacter orientalis, Saccharomyces cerevisiae, Lactobacillus paracasei and Acetobacter orientalis , Saccharomyces cerevisiae, Lactobacillus plantarum and Acetobacter orientalis , Saccharomyces cerevisiae , Lactobacillus acidophilus ) and Acetobacter orientalis , Saccharomyces cerevisiae , Lactobacillus kefiri and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus kefiranofaciens and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus helveticus and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactococcus lactis and Acetobacter lovaniensis ), brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus brevis and Acetobacter lovaniensis , brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus buchneri and Acetobacter lovaniensis , brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus casei subsp. Casei and Acetobacter lovaniensis , brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus casei subsp. Rhamnosus and Acetobacter lovaniensis , brewing yeast ( Saccharomyces cerevisiae ), Lactobacillus fermentum and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus mali and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus nagelli and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus paracasei and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus plantarum and Lactobacillus cerevisiae Lactobacillus plantarum and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus acidophilus and Acetobacter lovaniensis , Saccharomyces cerevisiae , Lactobacillus kefiri and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus kefiranofaciens and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus helveticus ) and Acetobacter aceti, Saccharomyces cerevisiae , Lactococcus lactis and Acetobacter aceti, Saccharomyces cerevisiae , Lactobacillus brevis and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus buchneri and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus casei subsp. Casei and Acetobacter aceti Acetobacter aceti ), Saccharomyces cerevisiae , Lactobacillus casei subsp. Rhamnosus and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus fermentum and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus mali and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus nagelli and Acetobacter aceti aceti ), Saccharomyces cerevisiae , Lactobacillus paracasei and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus plantarum and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus acidophilus and Acetobacter aceti , Saccharomyces cerevisiae , Lactobacillus kefiri and Acetobacter rasens , Saccharomyces cerevisiae ), Lactobacillus kefiranofaciens and Acetobacter rasens, Saccharomyces cerevisiae , Lactobacillus helveticus and Acetobacter rasens , Saccharomyces cerevisiae , Lactococcus lactis and Acetobacter rasens , Saccharomyces cerevisiae , Lactobacillus brevis and Acetobacter rasens , Saccharomyces cerevisiae , Lactobacillus bruvis and Acetobacter rasens Lactobacillus buchneri and Acetobacter rasens , Saccharomyces cerevisiae , Lactobacillus casei subsp. Casei and Acetobacter rasens , Saccharomyces cerevisiae , Lactobacillus casei subsp. Rhamnosus and Acetobacter rasens , Saccharomyces cerevisiae, Lactobacillus fermentum and Acetobacter rasens , Saccharomyces cerevisiae , Lactobacillus mali ) and Acetobacter rasens , Saccharomyces cerevisiae , Lactobacillus nagelli and Acetobacter rasens , Saccharomyces cerevisiae, Lactobacillus paracasei and Acetobacter rasens , Saccharomyces cerevisiae , Lactobacillus plantarum and Acetobacter rasens , or Saccharomyces cerevisiae , Lactobacillus acidophilus and Acetobacter rasens .

In these embodiments, taking the kefir bacteria comprising Kluyveromyces lactis as an example, the bacterial species combination can be listed as Kluyveromyces lactis , Lactobacillus kefiri and Acetobacter fabarum, Kluyveromyces lactis , Lactobacillus kefiranofaciens and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus helveticus and Acetobacter fabarum, Kluyveromyces lactis , Lactococcus lactis and Acetobacter fabarum. lactococcus lactis and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus brevis and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus buchneri and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus casei subsp. Casei and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus casei subsp. Rhamnosus ) and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus fermentum and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus mali and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus nagelli and Acetobacter fabarum, Kluyveromyces lactis , Lactobacillus paracasei and Acetobacter fabarum ), Kluyveromyces lactis , Lactobacillus plantarum and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus acidophilus and Acetobacter fabarum , Kluyveromyces lactis , Lactobacillus kefiri and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus kefiranofaciens and Acetobacter orientalis , Kluyveromyces lactis ), Lactobacillus helveticus , and Acetobacter orientalis , Kluyveromyces lactis , Lactococcus lactis , and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus brevis , and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus buchneri , and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus casei subsp. Casei ) and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus casei subsp. Rhamnosus and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus fermentum and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus mali and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus nagelli and Acetobacter orientalis ), Kluyveromyces lactis , Lactobacillus paracasei and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus plantarum and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus acidophilus and Acetobacter orientalis , Kluyveromyces lactis , Lactobacillus kefiri and Acetobacter lovaniensis , Kluyveromyces lactis ), Lactobacillus kefiranofaciens and Acetobacter lovaniensis, Kluyveromyces lactis, Lactobacillus helveticus and Acetobacter lovaniensis , Kluyveromyces lactis , Lactococcus lactis and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus brevis and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus buchneri ) and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus casei subsp. Casei and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus casei subsp. Rhamnosus and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus fermentum and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus mali and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus nagelli , and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus paracasei , and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus plantarum , and Acetobacter lovaniensis , Kluyveromyces lactis , Lactobacillus acidophilus , and Acetobacter lovaniensis , Kluyveromyces lactis ), Lactobacillus kefiri and Acetobacter aceti, Kluyveromyces lactis, Lactobacillus kefiranofaciens and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus helveticus and Acetobacter aceti , Kluyveromyces lactis , Lactococcus lactis and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus breve and Acetobacter aceti brevis and Acetobacter aceti, Kluyveromyces lactis , Lactobacillus buchneri and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus casei subsp. Casei and Acetobacter aceti, Kluyveromyces lactis , Lactobacillus casei subsp. Rhamnosus and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus fermentum ) and Acetobacter aceti, Kluyveromyces lactis , Lactobacillus mali and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus nagelli and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus paracasei and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus plantarum and Acetobacter aceti ), Kluyveromyces lactis , Lactobacillus acidophilus and Acetobacter aceti , Kluyveromyces lactis , Lactobacillus kefiri and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus kefiranofaciens and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus helveticus and Acetobacter rasens , Kluyveromyces lactis ), lactococcus lactis , and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus brevis , and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus buchneri , and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus casei subsp. Casei , and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus casei subsp. Rhamnosus ) and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus fermentum and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus mali and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus nagelli and Acetobacter rasens , Kluyveromyces lactis , Lactobacillus paracasei and Acetobacter rasens , Kluyveromyces Kluyveromyces lactis ), Lactobacillus plantarum , and Acetobacter rasens , or Kluyveromyces lactis , Lactobacillus acidophilus , and Acetobacter rasens .

In these embodiments, taking kefir bacteria including Kluyveromyces marxianus as an example, the bacterial species combination can be listed as Kluyveromyces marxianus , Lactobacillus kefiri and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus helveticus and Acetobacter fabarum . ), Kluyveromyces marxianus , lactococcus lactis , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus brevis , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus buchneri , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus casei subsp. Casei , and Acetobacter fabarum ), Kluyveromyces marxianus , Lactobacillus casei subsp. Rhamnosus , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus fermentum , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus mali , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus nagelli , and Acetobacter fabarum ), Kluyveromyces marxianus , Lactobacillus paracasei , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus plantarum , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus acidophilus , and Acetobacter fabarum , Kluyveromyces marxianus , Lactobacillus kefiri , and Acetobacter orientalis ), Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus helveticus and Acetobacter orientalis , Kluyveromyces marxianus , Lactococcus lactis and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus brevis and Acetobacter orientalis ), Kluyveromyces marxianus , Lactobacillus buchneri and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus casei subsp. Casei and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus casei subsp. Rhamnosus and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus fermentum and Acetobacter orientalis orientalis , Kluyveromyces marxianus , Lactobacillus mali , and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus nagelli , and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus paracasei , and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus plantarum , and Acetobacter orientalis ), Kluyveromyces marxianus , Lactobacillus acidophilus , and Acetobacter orientalis , Kluyveromyces marxianus , Lactobacillus kefiri , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus kefiranofaciens , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus helveticus , and Acetobacter lovaniensis ), Kluyveromyces marxianus , lactococcus lactis , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus brevis , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus buchneri , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus casei subsp. Casei , and Acetobacter lovaniensis ), Kluyveromyces marxianus , Lactobacillus casei subsp. Rhamnosus, and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus fermentum , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus mali , and Acetobacter lovaniensis, Kluyveromyces marxianus , Lactobacillus nagelli , and Acetobacter lovaniensis ), Kluyveromyces marxianus , Lactobacillus paracasei , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus plantarum , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus acidophilus , and Acetobacter lovaniensis , Kluyveromyces marxianus , Lactobacillus kefiri , and Acetobacter aceti ), Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus helveticus and Acetobacter aceti , Kluyveromyces marxianus , Lactococcus lactis and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus brevis and Acetobacter aceti ), Kluyveromyces marxianus , Lactobacillus buchneri and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus casei subsp. Casei and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus casei subsp. Rhamnosus and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus fermentum ) and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus mali and Acetobacter aceti , Kluyveromyces marxianus, Lactobacillus nagelli and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus paracasei and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus plantarum ) and Acetobacter aceti, Kluyveromyces marxianus , Lactobacillus acidophilus and Acetobacter aceti , Kluyveromyces marxianus , Lactobacillus kefiri and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus kefiranofaciens and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus helveticus ) and Acetobacter rasens , Kluyveromyces marxianus, Lactococcus lactis and Acetobacter rasens, Kluyveromyces marxianus , Lactobacillus brevis and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus buchneri and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus casei subsp. Casei and Acetobacter rasens ), Kluyveromyces marxianus , Lactobacillus casei subsp. Rhamnosus , and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus fermentum , and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus mali , and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus nagelli , and Acetobacter rasens ), Kluyveromyces marxianus , Lactobacillus paracasei , and Acetobacter rasens , Kluyveromyces marxianus , Lactobacillus plantarum , and Acetobacter rasens , or Kluyveromyces marxianus , Lactobacillus acidophilus , and Acetobacter rasens .

It should be noted that the above only partially lists the combinations of several species of complex bacterial species in probiotics to illustrate the implementation when the probiotics contain a combination of two, three or four or more different bacterial species, but is not intended to limit the implementation of the present invention.

Please refer to FIG. 1B , which illustrates a second embodiment of the method provided by the present invention, wherein the process steps are the same as those of the first embodiment, but the method further includes a temperature control treatment (S2), which includes placing the probiotics at a temperature-controlled temperature for a second time, wherein the temperature-controlled temperature is lower than room temperature, and the second time is greater than or equal to the first time.

Specifically, the temperature control treatment adopted in the present invention provides the probiotics with an adverse condition; under the adverse condition, the physiological mechanism of the probiotics will be stimulated to change, such as producing cold-adapted proteins or some secondary metabolites and enzymes. Specifically, exposure to a low temperature environment leads to a decrease in proteins related to energy metabolism activities.

In these embodiments, the probiotics are treated with temperature control to more effectively increase the subsequent vitamin D content, wherein the temperature control temperature is specifically a temperature less than or equal to the human body's basal temperature, which is generally defined as 36.1 to 37.5 degrees Celsius, which is the temperature exhibited in a normal activity state, such as static activity, and without external pathogen infection; in some preferred In an embodiment, the temperature control temperature includes a first temperature or a second temperature, the first temperature is less than or equal to the human body basal temperature, and the second temperature is less than the first temperature; in some specific embodiments, the first temperature is 30 to 40 degrees Celsius, and can be listed as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 degrees Celsius, and the second temperature is 0 to 24 degrees Celsius, can be listed as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 degrees Celsius; more preferably, the first temperature is 30 to 35 degrees Celsius or 35 to 40 degrees Celsius, specifically listed as 30, 31, 32, 33, 34, or 35 degrees Celsius. 5 degrees Celsius, or 35, 36, 37, 38, 39 or 40 degrees Celsius, the second temperature is 0 to 10 degrees Celsius or 10 to 24 degrees Celsius, specifically 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 degrees Celsius, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 degrees Celsius.

In these embodiments, in order to achieve the effect of increasing the vitamin D content by the temperature control treatment and to achieve the time condition for initiating changes in the physiological mechanism of the fungus, the second time is at least 60 minutes, preferably 60 to 120 minutes.

In the third embodiment of the method provided by the present invention, the process steps are the same as those of the first embodiment, but in order to achieve the purpose of irradiating the probiotics to increase the vitamin D content thereof, the irradiation treatment (S1) is specifically implemented by a specially designed irradiation device (100). Please refer to FIG. 2. The irradiation device (100) includes an outer shell (1) defining an irradiation space (11) inside thereof, a serving plate (2) disposed in the irradiation space (11), and an irradiation light source (3) disposed in the irradiation space (11), wherein the irradiation light source (3) is disposed at a distance of 100 Å from the serving plate (2). In addition, the outer shell (1) can be made of a reflective material or a partially reflective material, or can be made of a general material but coated with a reflective material. In addition, the serving plate (2) can be made of a reflective material or a transparent material. In a specific implementation, if the outer shell (1) is made of a reflective material, the serving plate (2) can be made of a transparent material. If the outer shell (1) is made of a general material, the serving plate (2) must be made of a reflective material. The probiotics are evenly spread on the serving plate (2), and the light from the irradiation light source (3) is reflected by the reflective material so that the probiotics are evenly irradiated.

In the fourth embodiment of the method provided by the present invention, the process steps are the same as the first embodiment, except that the irradiation light source irradiates a fungus, wherein the fungus includes an edible mushroom, such as Lentinula edodes , Volvariella volvacea , Agaricus bisporus , Hypsizygus tessulatus , Flammulina velutipes , Pleurotus eryngii , Auricularia auricula-judae, Agrocybe aegerita ( V. Brig. ) Singer or Ganoderma lucidum .

In addition, the following several embodiments are used to further demonstrate the practical application scope of the present invention, but are not intended to limit the scope of protection of the present invention. Generally, all modifications and improvements based on the spirit of the present invention are within the scope of protection declared by the present invention.

In the following examples, the kefir bacteria used are dried bacteria blocks, which include Kluyveromyces marxianus , Lactococcus lactis , Lactiplantibacillus plantarum and Lactobacillus casei . Before the temperature control treatment and irradiation treatment, the dried kefir bacteria blocks are washed with clean water and then centrifuged and drained at a low speed.

Embodiment 1

100 g of dried kefir bacteria were taken and placed on a plate in an illumination device. In Example 1, UV light and infrared light were used to illuminate the dried kefir bacteria to compare the effects of light sources of different wavelengths on the vitamin D content in the dried kefir bacteria. The vitamin D content was determined using a known vitamin D determination method. Please refer to Table 1 and FIG. 3A. After treatment, no vitamin D was detected in the dried kefir bacteria irradiated with red light, but the vitamin D content was detected in the dried kefir bacteria irradiated with ultraviolet light, which increased from 0μg/100g to 23μg/100g. This shows that ultraviolet light can effectively promote the production of vitamin D in the dried kefir bacteria. Table 1 Vitamin D production (μg/100g) Infrared light 0 UV light twenty three

Embodiment 2

100 grams of dried kefir bacteria were taken and placed in a low temperature environment of 5 to 15 degrees Celsius for 60 to 120 minutes, and then placed on a plate in an irradiation device. In Example 2, the dried kefir bacteria were irradiated with different light source intensities to compare the effects of different UV light intensities on the amount of vitamin D produced in the dried kefir bacteria, and the vitamin D content was measured using a known vitamin D determination method. Please refer to FIG. 3B and Table 2. After the temperature control treatment and irradiation treatment, the amount of vitamin D produced in the dried kefir bacteria treated at any UV irradiation intensity was measured, and at 0.2 mW/cm The vitamin D production of dried Kefir bacteria increased from 3μg/100g to 15μg/100g at an irradiation intensity of ² , reaching a peak at an irradiation intensity of 0.5mW/ ^cm2 , but gradually decreased with increasing irradiation intensity. Table 2 Irradiation intensity (mW/cm ² ) Vitamin D production (μg/100g) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 Kefir dried bacteria 0 3 15 17 20 twenty three twenty two twenty two twenty two 20 15 14 11

Embodiment 3

100 g of dried Kefir bacteria were placed in a low temperature environment of 5 to 15 degrees Celsius for 60 to 120 minutes, and then placed on a plate in a lighting device. In Example 3, the light was irradiated at 0.4 to 0.6 mW/cm ² , and irradiation treatment was carried out at different irradiation distances to compare the effects of different irradiation distances on the vitamin D content in kefir dried mushrooms, and the production amount was measured using the known vitamin D determination method; please refer to Figure 3C and Table 3. After temperature control treatment and irradiation treatment, the production amount of vitamin D was measured in kefir dried mushrooms treated at any irradiation distance greater than 1 cm, and at an irradiation distance of 4 to 6 cm, the average vitamin D production of kefir dried mushrooms reached 22.7±0.6 μg/100g, but the vitamin D production of shiitake mushrooms or combined mushrooms gradually decreased with the increase of irradiation distance. Table 3 Irradiation distance (cm) Vitamin D production (μg/100g) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Kefir dried bacteria 0 3 20 twenty three twenty three twenty two 20 15 11 10

Embodiment 4

100 g of dried Kefir bacteria were placed in a low temperature environment of 5 to 15 degrees Celsius for 60 to 120 minutes, and then placed on a plate in a lighting device. In Example 4, the light was irradiated at 0.4 to 0.6 mW/cm ² , the irradiation distance was set to 4 to 6 cm, and the irradiation treatment was carried out at different irradiation times to compare the effects of different irradiation times on the production of vitamin D in the dried kefir bacteria, and the production was measured using the known vitamin D determination method; please refer to Figure 3D and Table 4. After temperature control treatment and irradiation treatment, the production of vitamin D in the dried kefir bacteria began to be measured at least 20 minutes after irradiation; after 60 to 120 minutes of irradiation, the average vitamin D content of the dried kefir bacteria reached 25.0±1.2 μg/100g, and under the irradiation condition of more than 60 minutes, the vitamin D content gradually increased with the increase of irradiation time, indicating that the longer the irradiation time, the greater the production of vitamin D. Table 4 Irradiation time (cm) Vitamin D production (μg/100g) 0 10 20 30 40 50 60 70 80 90 100 110 120 Kefir dried bacteria 0 0 2 10 14 19 twenty three twenty four 26 25 26 26 25

Embodiment 5

100 g of dried Kefir bacteria were placed in a temperature range of 5 to 40 degrees Celsius for 60 to 120 minutes, and then placed on a tray in a lighting device for light treatment. In Example 5, the temperature was 0.4 to 0.6 mW/cm ² , the irradiation distance was set to 4 to 6 cm and the irradiation time was at least 60 minutes to compare the effect of static treatment under different temperature conditions on the production of vitamin D in the dried Kefir bacteria. The production was measured using the known vitamin D determination method. Please refer to Figure 3E and Table 5. After being treated at a low temperature of 5°C for at least 60 to 120 minutes, the production of vitamin D in the dried Kefir bacteria treated with light began to be measured, and the average vitamin D production reached 21.0±2.0 μg/100g, and when the static temperature is greater than 15℃, the amount of vitamin D produced gradually decreases with the increase of temperature, but when the static temperature reaches 25℃ to 35℃, the amount of vitamin D produced increases with the increase of temperature. Table 5 Temperature condition (℃) Vitamin D production (μg/100g) 0 5 10 15 20 25 30 35 40 Kefir dried bacteria 0 19 twenty three twenty one 14 15 18 twenty three 11

The method provided by the present invention only requires a photocatalytic method to promote the conversion mechanism of the precursor of vitamin D in kefir bacteria. The synthesized vitamin D can be further purified and extracted, or added to food, health care products and other applications. The method is simple to prepare and eliminates the uncontrollable factors involved in the fermentation culture or extraction process from animal sources in the known technology, thereby ensuring the stability and safety of the product.

The method provided by the present invention is combined with temperature-controlled treatment of probiotics to increase the yield of vitamin D, and can be used in the food industry to manufacture products rich in vitamin D, so as to increase the vitamin D intake of consumers, thereby reducing the risk of osteoporosis, enhancing immune regulation, and preventing cancer, thereby improving the health effects of the product and promoting its economic benefits.

S0: Temperature control treatment S1: Irradiation treatment 100: Illumination device 1: Housing 11: Illumination space 2: Plate 3: Illumination light source

FIG. 1A is a first embodiment of the method for producing vitamin D provided by the present invention; FIG. 1B is a second embodiment of the method for producing vitamin D provided by the present invention; FIG. 2 is a cross-sectional view of the illumination device; FIG. 3A is a bar graph for illustrating the amount of vitamin D generated by different light sources in Example 1; and FIG. 3B to 3E are a series of line graphs for illustrating the amount of vitamin D generated under different parameters in Examples 2 to 5.

S1: Irradiation treatment

Claims (8)

A method for producing vitamin D content, comprising: an irradiation treatment: irradiating a probiotic with an irradiation light source for a first time to obtain vitamin D, wherein the irradiation light source includes natural light or ultraviolet light, and the probiotic includes at least a yeast, a lactic acid bacterium and an acetic acid bacterium; and a temperature control treatment, before the irradiation treatment: placing the probiotic at a temperature-controlled temperature for a second time, the temperature-controlled temperature is between 0 degrees Celsius and 24 degrees Celsius but not 0 degrees Celsius, and the second time is greater than or equal to the first time. The method as described in claim 1, wherein the yeast comprises Saccharomyces cerevisiae , Kluyveromyces lactis , Kluyveromyces marxianus , Pichia membranifaciens , Pichia kudriavzevii , Lanchancea fermentati , Lanchancea meyercii , Kazachstania aerobia or Kazachstania unispora or any combination of two or more thereof. The method as described in claim 1, wherein the lactic acid bacteria include Lactobacillus kefiri , Lactobacillus kefiranofaciens , Lactobacillus helveticus , Lactococcus lactis , Lactobacillus brevis , Lactobacillus buchneri , Lactobacillus casei subsp. casei , Lactobacillus casei subsp. rhamnosus, Lactobacillus fermentum, Lactobacillus mali , Lactobacillus nagelli, Lactobacillus paracasei , Lactobacillus spp. Lactobacillus paracasei ), Lactobacillus plantarum , Lactobacillus acidophilus , or any combination of two or more thereof. The method as described in claim 1, wherein the acetic acid bacteria comprises Acetobacter fabarum , Acetobacter orientalis , Acetobacter lovaniensis , Acetobacter aceti, Acetobacter rasens or any combination of two or more thereof. The method as described in claim 1, wherein the probiotics include a yeast, a lactic acid bacterium and an acetic acid bacterium, wherein the yeast includes Saccharomyces cerevisiae , Kluyveromyces lactis , Kluyveromyces marxianus , Pichia membranifaciens , Pichia kudriavzevii , Lanchancea fermentati , Lanchancea meyercii , Kazachstania aerobia or Kazachstania unispora , and the lactic acid bacteria include Lactobacillus kefiri , Lactobacillus kumis kefiranofaciens ), Lactobacillus helveticus , Lactococcus lactis , Lactobacillus brevis , Lactobacillus buchneri , Lactobacillus casei subsp.casei , Lactobacillus casei subsp.rhamnosus , Lactobacillus fermentum , Lactobacillus mali , Lactobacillus nagelli , Lactobacillus paracasei , Lactobacillus plantarum , Lactobacillus acidophilus ) or any combination of two or more thereof, wherein the acetic acid bacteria includes Acetobacter fabarum, Acetobacter orientalis, Acetobacter lovaniensis, Acetobacter aceti or Acetobacter rasens. The method as described in claim 1, wherein the probiotics include a yeast, a lactic acid bacterium, and an acetic acid bacterium, wherein the yeast is Kluyveromyces lactis or Kluyveromyces marxianus , the lactic acid bacterium is Lactococcus lactis , Lactobacillus casei subsp. casei, Lactobacillus plantarum or any combination of two or more thereof, and the acetic acid bacterium is Acetobacter fabarum or Acetobacter orientalis. The method as described in claim 1, wherein the irradiation treatment further comprises: before the irradiation light source irradiates the probiotics, the probiotics are placed at a distance from the irradiation light source, the irradiation distance being 2 to 10 centimeters. The method as described in claim 1, wherein the first time is at least 30 minutes and the second time is 60 to 120 minutes.