CN114181842B - Saccharomycopsis fibuligera cx-3 strain for converting anthocyanin to produce protocatechuic acid and application thereof - Google Patents
Saccharomycopsis fibuligera cx-3 strain for converting anthocyanin to produce protocatechuic acid and application thereof Download PDFInfo
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Abstract
The invention discloses a cyst membrane-covering yeast (Saccharomyces fibuligera) cx-3 strain for converting anthocyanin to produce protocatechuic acid and application thereof, wherein the strain is preserved in Guangdong province microorganism strain preservation center at 2021, 9, 16 days, and the preservation number is GDMCC NO:61938. the invention discloses a capsule-covering membrane yeast cx-3 strain which can convert anthocyanin in the sorbus margaritae fermentation process and produce protocatechuic acid, can improve the bioavailability of the anthocyanin in the sorbus margaritae, and greatly improves the absorption and utilization of anthocyanin substances in human intestinal tracts. The strain is used in a fermentation process of the sorbus pohuashanensis ferment and the mulberry ferment, and protocatechuic acid produced by the strain can enable the ferment to have better health care value and probiotic property. The invention provides excellent strains for the food industry, and has good application value in the fermentation process of the sorbus pohuashanensis ferment.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly, relates to a strain of Saccharomycopsis fibuligera cx-3 for converting anthocyanin into protocatechuic acid and application thereof.
Background
Saccharomycopsis fibuligera is a yeast capable of producing hyphae and ascospores, also called Saccharomycopsis fibuligera and Ascomycopsis fibuligera, which is a yeast-like cell formed by budding and has typical biphasic property. Because of its strong starch decomposing ability, it is widely distributed in the matrix containing a large amount of starch, and is also the main fragrant fungus and functional strain in the fen-flavor liquor distiller's yeast.
Anthocyanins (anthocyanidins) are a collective term for a large group of water-soluble natural pigments, which are ubiquitous in the vacuolar tissues of plant flowers, fruits, stems and leaves, etc. Anthocyanins are also polyphenols that are ubiquitous in ordinary dietary nutrition, with the chemical structure being flavonoids (flavanoids); many studies at present find that the main flavonoid compounds in the metabolic pathway of the human body are the conditions that the flavonoid compounds cannot be degraded by digestive juice and are directly absorbed, and need to be converted into simple phenolic acid by intestinal flora secretase to be utilized or discharged out of the body. With the continuous research on functional ingredients of health foods in recent years, the result of the research on bioavailability of anthocyanin in metabolic pathways in human bodies is not ideal as expected. It is generally considered that anthocyanins enter blood vessels in stomach cells after entering the digestive system, and anthocyanins entering blood circulation in stomach parietal cells can be detected for a short time, but even if anthocyanins can be stably present in an environment where the pH of gastric acid =2, the absorption rate in the stomach is very low. In the known method for converting protocatechuic acid by using a microorganism whole cell to catalyze natural anthocyanin and application thereof, after a bacterial strain is cultured for 48 hours in an MRS culture medium added with anthocyanin extract in a certain proportion externally used in vitro, HPLC (high performance liquid chromatography) detection finds that the protocatechuic acid yield in a culture solution can reach 3.3mg/L, and a bacterial functional strain capable of biologically converting purple sweet potato anthocyanin into protocatechuic acid is found (Chinese patent laid-open publication No. 201710212720.8). The functional fungus strain is also a microbial resource for biotransformation of anthocyanin, but the anthocyanin can be found to be biotransformed into protocatechuic acid in the enzymatic fermentation process of the sorbus pohuashanensis. Even so, microbial resources for biotransformation of anthocyanins to protocatechuic acids, which are similarly so effective, are comparatively small.
Although the saccharomyces cerevisiae belongs to aroma-producing yeasts, the strain is rarely reported to be used for converting anthocyanin to produce protocatechuic acid, and the functional strain of the saccharomyces cerevisiae capable of improving the probiotic function of sorbus pohuashanensis enzyme has never been reported.
Disclosure of Invention
The invention aims to provide a bacterial strain of saccharomyces cerevisiae fibuligera (cx-3) for producing protocatechuic acid by converting anthocyanin.
The invention provides application of the envelope-covering yeast cx-3 strain.
The invention provides a sorbus pohuashanensis enzyme fermentation method.
The above purpose of the invention is realized by the following technical scheme: a sacculus-coated yeast (Saccharomyces fibuligera) cx-3 strain for converting anthocyanin to produce protocatechuic acid is preserved in Guangdong province microbial culture collection center (GDMCC) at 9, 16 months in 2021, and the preservation address is No. 59 building of Michelia Tokyo 100, guangzhou, guangdong province, with the preservation number being GDMCC NO:61938.
the invention discovers that the cyst-coating yeast cx-3 strain separated from the highland barley alcohol koji can convert anthocyanin into protocatechuic acid through metabolism in the ferment fermentation process. Researches show that the strain can convert anthocyanin in the sorbus margaritae fermentation process and produce protocatechuic acid, can improve the bioavailability of the anthocyanin in the sorbus margaritae, and greatly improve the absorption and utilization of anthocyanin substances in human intestinal tracts. The strain is used in a fermentation process of the sorbus pohuashanensis ferment and the mulberry ferment, and protocatechuic acid produced by the strain can enable the ferment to have better health care value and probiotic property. The invention provides excellent strains for the food industry and has good application value in the fermentation process of the sorbus pohuashanensis ferment.
The invention provides application of the envelope-buckled membrane-covered yeast cx-3 strain in ferment fermentation of sorbus pohuashanensis.
Preferably, the cyst-coated yeast cx-3 strain is inoculated into the homogenized sorbus pohuashanensis according to the inoculation amount of 0.5-2 wt%, sterile water with the weight ratio of 3.
The invention provides application of the cyst-coating yeast cx-3 strain in fermentation of mulberry ferment.
Preferably, the cyst-coating yeast cx-3 strain is inoculated into the homogenized mulberry fruit according to the inoculation amount of 0.5-2 wt%, sterile water with the weight ratio of 3.
The invention provides a sorbus pohuashanensis enzyme fermentation method, which comprises the following steps: the mountain ash black fruit is used as a substrate, and the above-mentioned strain of the envelope-covering yeast cx-3 is adopted for fermentation.
Preferably, the yield of protocatechuic acid in the sorbus pohuashanensis enzyme is up to 60 mu g/ml.
The invention provides a mulberry enzyme fermentation method, which comprises the following steps: and (3) fermenting by using the cx-3 strain and taking the mulberry purple fruits as substrates.
Preferably, the yield of protocatechuic acid in the mulberry enzyme is up to 40-50 mu g/ml.
The invention has the following beneficial effects:
the invention provides a strain of saccharomyces capilalis muligera (Saccharomyces fibuligera) cx-3 for converting anthocyanin into protocatechuic acid, which is inoculated into homogenized sorbus pohuashanensis fruits, sterile water is added, and the strain is fermented in a sealing way, so that the anthocyanin in the sorbus pohuashanensis is converted by the strain, and the yield of protocatechuic acid can reach about 60 mu g/ml. The characteristic that the strain converts anthocyanin to produce protocatechuic acid can be used for fermentation of sorbus pohuashanensis enzymes, the health-care value and the probiotic characteristic of sorbus pohuashanensis enzymes can be increased, the strain can also be used for fermentation of other fruit enzymes rich in anthocyanin, the microbial strain capable of converting anthocyanin to protocatechuic acid is provided, and the strain has good application value.
Drawings
FIG. 1 is a HPLC peak chart of the strain plus bacteria fermentation group and a standard protocatechuic acid; note: peak 1: protocatechuic acid standard peak, peak 2: a cx-3 strain fermentation group peak;
FIG. 2 is the HPLC peak chart of the blank fermentation group and the standard protocatechuic acid, note: peak 1: protocatechuic acid standard peak;
FIG. 3 is a HPLC peak chart of proto-fruit juice group and protocatechuic acid as a standard; note: peak 1: protocatechuic acid standard peak;
FIG. 4 is a bar graph showing the change in the concentration of protocatechuic acid in the Sorbus commixta ferment fermented by the strain.
Detailed Description
The invention is further described with reference to the drawings and specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 fermentation preparation of Sorbus pohuashanensis enzyme and measurement of protocatechuic acid production amount
Weighing 300g of mountain ash black fruit, cleaning, naturally drying, and crushing the mountain ash black fruit into fine and smooth shapes by using a homogenizing tool sterilized by boiling water for later use.
This fermentation was carried out using a strain of Saccharomyces cerevisiae cx-3 which has the ability to convert anthocyanins to protocatechins. Inoculating the activated yeast of two generations into 50ml of MRS liquid culture medium according to the inoculation amount of 0.5-2% (w/w, weight ratio), and carrying out shake culture at 37 ℃ for 24h. Centrifuging at 4000r/min for 10min, removing supernatant, washing thallus with sterile water, centrifuging, removing supernatant, and precipitating thallus for use.
The mountain ash pomace mixed juice is added with a certain amount of sterile water according to the ratio ww of 3 (w/w, weight ratio), and then respectively set into a sugar-containing group and a sugar-free group. Adding 2% (w/w, weight ratio) of white granulated sugar into the sugar adding group, adding bacterial sludge according to the inoculation amount of 0.5-2% (w/w, weight ratio), stirring uniformly, and sealing with a fermentation tank. Meanwhile, preparing a blank fermentation group without adding bacteria, and putting the blank fermentation group in a 25-degree incubator together for fermentation for 20-25 d.
10ml of fermentation broth after 25 days of fermentation was aspirated. After dilution of the fermentation broth with 5% (v/v, volume ratio) acetonitrile water, it was filtered through a 0.22 μm filter and the protocatechuic acid concentration was determined by HPLC using the following procedure: protocatechuic acid was detected at 254 nm. HPLC detection conditions: column Merck (Germany) Purospher STAR LP RP-18 reversed-phase column (250 mm. Times.4.6 mm,5 μm), column temperature 30 ℃, detector DAD, mobile phase A: acetonitrile; mobile phase B:1% formic acid water (pH = 2.1), binary gradient elution procedure: keeping the B phase at 94% for 0-4 min; 4-15 min, reducing the B phase 95% to 70%; keeping the phase B for 16-20 min at 70%; the phase B rises from 70% to 94% in 20-25 min, and the LC collection time is 0-20 min. The flow rate was 1.0ml/min, and 20. Mu.L of sample was injected.
The HPLC detection results of the samples of each group are shown in the figure 1-figure 4.
The protocatechuic acid in the supernatant was detected, and it was found that the protocatechuic acid in the protofruit juice group and the blank fermentation group showed no peak at the peak appearance time of the standard, while the peak shape of the protocatechuic acid in the catalpa bungei fermentation group of the cx-3 strain was significantly increased, and the protocatechuic acid concentration was 62.6383 μ g/ml (fig. 1-4). The microbial interaction utilizes the anthocyanin rich in the sorbus pohuashanensis to be converted into protocatechuic acid in the fermentation process.
In the fermentation process of the sorbus pohuashanensis enzyme, the cx-3 strain can convert anthocyanin to produce protocatechuic acid, so that feasibility is established for the application of the sorbus pohuashanensis enzyme in various fields, besides the anthocyanin can be converted to produce protocatechuic acid, the original taste of the sorbus pohuashanensis can be removed by degrading tannin, and the like, so that the sorbus pohuashanensis enzyme is possibly applied to the fermentation of the sorbus poshanensis enzyme. The cx-3 strain is inoculated into the sorbus pohuashanensis juice to become a dominant strain fermentation process in the sorbus pohuashanensis enzyme fermentation process, so that the bioavailability of anthocyanin in the sorbus pohuashanensis can be improved, the taste of the sorbus pohuashanensis enzyme can be obviously improved, and the results further provide basis for the application of the strain in sorbus pohuashanensis fermentation. Therefore, the health value of the sorbus pohuashanensis ferment can be purposefully improved by controlling the inoculation amount and the fermentation conditions of the cx-3 strain in the subsequent research.
Example 2 fermentation preparation of Mulberry enzyme and measurement of protocatechuic acid production thereof
Weighing 100g of mulberry, cleaning, naturally drying, and simply homogenizing the mulberry by using a homogenizing tool disinfected by boiling water for later use.
Fermentation was performed using a strain of Saccharomyces cerevisiae fibuligera (Cx-3) having the ability to convert anthocyanins to protocatechuic acids. Inoculating the activated yeast of two generations into 50ml of MRS liquid culture medium according to the inoculation amount of 0.5-2% (w/w, weight ratio), and carrying out shake culture at 37 ℃ for 24h. Centrifuging at 4000r/min for 10min, removing supernatant, washing thallus with sterile water, centrifuging, removing supernatant, and precipitating thallus for use.
Mixing the mulberry pomace with the juice, and mixing the juice with the mulberry pomace according to the ratio of 3:1 (w/w, weight ratio) of sterile water, 2-4% (w/w, weight ratio) of white granulated sugar, bacterial sludge according to the inoculum size of 0.5-2% (w/w, weight ratio), stirring uniformly and sealing with a fermentation tank. Fermenting for 20-25 days in a 25 ℃ incubator.
HPLC detection is respectively carried out on the original fruit juice group (unfermented group), the blank fermentation group and the cx-3 strain fermentation group. After dilution of the sample with 5% (v/v, vol) acetonitrile, it was filtered through a 0.22 μm filter and the protocatechuic acid concentration was determined by HPLC using the following procedure: protocatechuic acid was detected at 254 nm. HPLC detection conditions: column Merck (Germany) Purospher STAR LP RP-18 reversed-phase column (250 mm. Times.4.6 mm,5 μm), column temperature 30 ℃, detector DAD, mobile phase A: acetonitrile; and (3) mobile phase B:1% formic acid water (pH = 2.1), binary gradient elution procedure: keeping the B phase at 94% for 0-4 min; 4-15 min, reducing the B phase 95% to 70%; keeping the phase B at 70% for 16-20 min; the phase B rises from 70% to 94% in 20-25 min, and the LC collection time is 0-20 min. The flow rate was 1.0ml/min, and 20. Mu.L of sample was injected.
Through detecting protocatechuic acid in the supernatant, the protocatechuic acid content in the protocatechuic fruit juice group is low and nearly absent, while the peak shape of the protocatechuic acid in the mulberry fermentation group of the cx-3 bacterial strain is obviously increased, and the protocatechuic acid concentration reaches about 50 mu g/ml.
The characteristic that the cx-3 strain can convert anthocyanin to produce protocatechuic acid provides good feasibility for the strain to be applied to fermentation products of anthocyanin-rich fruit juice. The cx-3 bacterial strain is inoculated into the mixed juice of the mulberry pomace to become a dominant strain leading fermentation process in a mulberry enzyme fermentation process, so that the bioavailability of anthocyanin in the mulberry can be improved, the health care value of the mulberry enzyme is improved, and the results further provide a basis for the application of the bacterial strain in the mulberry fermentation. Therefore, the health care value of the mulberry enzyme can be purposefully improved by controlling the inoculation amount and the fermentation condition of the cx-3 strain in the subsequent research.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A sacculus-coated yeast (Saccharomyces fibuligera) cx-3 strain for converting anthocyanin to produce protocatechuic acid is characterized in that the strain is stored in the microbial strain collection center (GDMCC) in Guangdong province in 9 and 16 months 2021, the storage address is No. 59 building 5 building of Michelia Tokyo 100, guangzhou, guangdong province, and the storage number is GDMCC NO:61938.
2. the use of the envelope-buckled yeast cx-3 strain according to claim 1 in enzymatic fermentation of Sorbus pohuashanensis.
3. The application of claim 2, wherein the envelope-covering yeast cx-3 strain is inoculated into the homogenized Sorbus pohuashanensis at an inoculation amount of 0.5-2 wt%, sterile water is added in an amount of 3:1 by weight, the mixture is sealed and fermented for 20-25 days, the anthocyanin in the Sorbus pohuashanensis is converted by the envelope-covering yeast cx-3 strain, and the protocatechuic acid yield reaches 50-60 [ mu ] g/ml.
4. The use of the saccharomyces cerevisiae cx-3 strain of claim 1 in mulberry enzyme fermentation.
5. The application of the strain as claimed in claim 4, wherein the strain of the Saccharum sinensis Ensiformis yeast cx-3 is inoculated into the homogenized mulberry fruit according to the inoculation amount of 0.5-2 wt%, and 3:1 weight proportion of sterile water, sealing and fermenting for 20-25 days, converting anthocyanin in the mulberry by the strain, and enabling the protocatechuic acid yield to reach 40-50 mu g/ml.
6. A mountain ash enzyme fermentation method is characterized by comprising the following steps: the catalpa bungei black fruit is used as a substrate, and the strain of the Saccharomycopsis fibuligera cx-3 as claimed in claim 1 is used for fermentation.
7. The fermentation method according to claim 6, wherein the yield of protocatechuic acid in the Sorbus commixta ferment is up to 60 μ g/ml.
8. A mulberry enzyme fermentation method is characterized by comprising the following steps: fermenting with the cx-3 strain of claim 1 using mulberry purple fruit as a substrate.
9. The method of claim 8, wherein the yield of protocatechuic acid in the mulberry enzyme is up to 40-50 μ g/ml.
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