CN111621428A - Salt-tolerant rhodotorula mucilaginosa strain for high yield of phenethyl alcohol and application thereof - Google Patents
Salt-tolerant rhodotorula mucilaginosa strain for high yield of phenethyl alcohol and application thereof Download PDFInfo
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Abstract
The invention provides a salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol and application thereof. The strain has a preservation number of CGMCC No.19008, and is preserved in China general microbiological culture Collection Center (CCM) in 2019, 11 and 26 months. The strain has the characteristics of salt tolerance, good growth stability and capability of generating rich sauce flavor and mellow flavor, the content of active ingredients such as phenethyl alcohol, furanone, maltol, 2, 6-dimethyl pyrazine and the like in volatile substances is obviously increased, the strain can be used for fermenting high-salt dilute soy sauce, and the flavor and the quality of soy sauce products are improved.
Description
Technical Field
The invention belongs to the technical field of food fermentation processing, and particularly relates to a salt-tolerant Rhodotorula mucilaginosa strain (Rhodotorula mucor) for high yield of phenethyl alcohol and application thereof.
Background
Soy sauce is one of traditional fermented foods and has become a necessary seasoning for Chinese households. The unique color, smell and taste of the health-care beer can not separate from the interaction of different microorganisms in the brewing process, and simultaneously, the health-care beer also contains rich nutrient components and has certain health-care effect. In the 20 s of the 19 th century, basic indexes such as ammonia-base state nitrogen in soy sauce are more important, but with the improvement of living standard, consumers have higher requirements on color, aroma and taste of soy sauce, and high-quality soy sauce with good taste and flavor is gradually pursued, so that more and more researchers pay attention to flavor research of soy sauce, and the flavor of soy sauce is closely related to yeast participating in soy sauce fermentation.
Currently, many researchers are concerned with the mainstream salt-tolerant yeasts in soy sauce, such as zygosaccharomyces rouxii, and are less concerned with the non-mainstream yeasts related to flavor in soy sauce, such as candida, rhodotorula mucilaginosa and the like. The rhodotorula mucilaginosa is a marine microorganism, has strong capability of producing oil, has wide research field for the rhodotorula mucilaginosa, covers a mechanism for producing carotenoid, the antioxidant activity of extracellular polysaccharide and the separation and extraction of astaxanthin. Some researchers add the rhodotorula mucilaginosa and the saccharomyces cerevisiae into the white spirit fermentation to discuss the aroma enhancement effect, and find that the rhodotorula mucilaginosa is beneficial to improving the contents of C6-compounds, fatty acid ethyl esters, fatty acids and phenylethyl. However, the research on the strain in the soy sauce is less, and no one applies the strain to the high-salt dilute soy sauce brewing process to research the influence of the strain on the flavor components of the soy sauce.
In the research of soy sauce flavor components, the phenethyl alcohol has unique rose fragrance and is a key fragrance active substance of the soy sauce. Early studies found that Candida, zygosaccharomyces, saccharomyces cerevisiae and the like have certain effects on the production of phenethyl alcohol, such as those skilled in the courage and the like (climbing, new and honour, liudan, and the like. identification of 2-phenethyl alcohol producing yeast and application thereof in soy sauce fermentation [ J ]. food science 2019,40(06): 217-. Wu Yaman (Wu Yaman, Soy sauce, Saccharomyces rouxii S, S3-2 key flavor metabolite contrast research [ D ]. Tianjin: Tianjin science and technology university, 2015.) and Ruiying (Ruiyinging, Zhengjia, He Fei, etc.. salt tolerant lactic acid bacteria and yeast strengthening technology on the quality of high-salt dilute fermentation soy sauce mash [ J ]. food and fermentation industry, 2013,39(2):1-6.) and other researches find that Zygosaccharomyces rouxii and compounds such as 2-phenethyl alcohol in soy sauce are in close relation with each other in metabolic synthesis, and can improve the concentration of 2-phenethyl alcohol in soy sauce mash. Von Jie (Von Jie. Candida antarctica aroma production mechanism and influence on soy sauce flavor [ D ]. stannless: university of south Jiangnan, 2012.) reports that 1 strain of high-salt-resistant aroma-enhancing yeast Candida antarctica (Candidaetchelsii) can synthesize 2-phenylethyl alcohol in the soy sauce fermentation process. However, there is no report on the application of rhodotorula mucilaginosa in soy sauce for improving flavor.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol.
The invention aims to provide a salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol. The strain can grow and propagate in a high-salt environment for brewing high-salt dilute soy sauce, can generate higher-content phenethyl alcohol, generates soy sauce flavor with sauce fragrance and mellow fragrance, and improves the flavor quality of the soy sauce. The strain was identified as Rhodotorula mucilaginosa (Rhodotorula mucor) according to its morphological characteristics, growth characteristics and 18S rDNA gene sequence.
The salt-tolerant rhodotorula mucilaginosa strain for high yield of phenethyl alcohol is named as Rhodotorula micilaginosa with the preservation number of CGMCC No.19008, is preserved in the general microbiological center of China Committee for culture Collection of microorganisms in 11 months and 26 days in 2019, and is addressed to the institute of microbiology of Zhongkoyao institute No. 3 of Navy, Naja, Beijing.
The salt-tolerant rhodotorula mucilaginosa strain capable of highly producing phenethyl alcohol is a rhodotorula mucilaginosa strain capable of increasing the fragrance of soy sauce. The strain can be propagated and fermented in a high-salt environment for brewing high-salt dilute soy sauce, and can improve the flavor of the soy sauce.
The strain has the characteristics of salt tolerance, good growth stability and capability of generating rich sauce flavor and mellow flavor, the content of active ingredients such as phenethyl alcohol, furanone, maltol, 2, 6-dimethyl pyrazine and the like in volatile substances is obviously increased, the strain can be used for fermenting high-salt dilute soy sauce, and the flavor and the quality of soy sauce products are improved.
The purpose of the invention is realized by at least one of the following technical solutions.
The salt-tolerant Rhodotorula mucilaginosa strain (Rhodotorula mucor) for high yield of phenethyl alcohol provided by the invention has the following morphological characteristics: the colony is round, the diameter is 1mm, the colony is orange, and the surface is moist, smooth and raised.
According to the salt tolerance experiment result, the salt tolerance range (by sodium chloride NaCl tolerance concentration) is 0-20%.
According to the temperature growth experiment result, the most suitable growth temperature of the salt-tolerant Rhodotorula mucilaginosa strain (Rhodotorula mucor) for high yield of phenethyl alcohol provided by the invention is 35 ℃.
According to the pH growth experiment result, the most suitable growth pH of the salt-tolerant rhodotorula mucilaginosa (Rhodotorula mucilaginosa) strain for high yield of phenethyl alcohol is 5.
The salt-tolerant Rhodotorula mucilaginosa strain (Rhodotorula mucor) capable of producing phenethyl alcohol with high yield can be applied to soy sauce fermentation.
The salt-tolerant Rhodotorula mucilaginosa (Rhodotorula mucor) strain for high yield of phenethyl alcohol provided by the invention is added into volatile substances generated by fermentation in soy sauce, has higher phenethyl alcohol content and shows good sauce aroma and mellow flavor characteristics.
The application of the salt-tolerant rhodotorula mucilaginosa strain for high yield of phenethyl alcohol in soy sauce fermentation comprises the following steps:
(1) preparing salt-tolerant rhodotorula mucilaginosa strain liquid for high yield of phenethyl alcohol: inoculating the salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield to a culture medium for activation culture, repeating the operation for three times (passage for 3 times) to obtain a cultured bacterial liquid, centrifuging to obtain a precipitate, washing with sterile normal saline, and suspending in the sterile normal saline to obtain the salt-tolerant rhodotorula mucilaginosa bacterial liquid;
(2) preparing Daqu in the preparation process of high-salt dilute soy sauce: adding soybeans into water, soaking, taking out to obtain soaked soybeans, placing the soaked soybeans in an autoclave for high-pressure sterilization, cooling to room temperature to obtain cooked soybeans, and uniformly mixing the cooked soybeans, flour and yeast to obtain yeast;
(3) adding salt-tolerant rhodotorula mucilaginosa strains for high yield of phenethyl alcohol: putting the yeast in the step (2) into an incubator for solid culture to obtain finished yeast; and (2) uniformly mixing the finished koji with saline water to obtain sauce mash, carrying out primary natural fermentation treatment on the sauce mash, then adding the salt-tolerant rhodotorula mucilaginosa bacterial liquid obtained in the step (1), carrying out secondary natural fermentation treatment, sterilizing, filtering and taking filtrate to obtain the soy sauce.
Further, the culture medium in the step (1) is one of a wort liquid culture medium and a YPD liquid culture medium; the concentration of the salt-tolerant rhodotorula mucilaginosa bacterial liquid is 105-106CFU/mL。
Further, the temperature of the centrifugal sediment taking in the step (1) is 20 ℃, the rotating speed of the centrifugal sediment taking is 3000r/min, and the time of the centrifugal sediment taking is 10 min.
The salt-tolerant rhodotorula mucilaginosa bacterial liquid obtained in the step (1) can be stored in a refrigerator at 4 ℃ and used within 24 hours.
Further, the soaking treatment time in the step (2) is 6-8 h; the temperature of the high-pressure sterilization treatment is 121 ℃, and the time of the high-pressure sterilization treatment is 10-15 min.
Further, the mass ratio of the soybeans to the flour in the step (2) is 4:1 in terms of dry weight; the yeast extract is Aspergillus oryzae; according to dry weight, the quality of the yeast extract is 0.05-2% of the quality of the flour and the soybean.
Further, the total time of the solid state culture in the step (3) is 40-50 h; the solid state culture comprises: culturing at 30 deg.C for 20-25h, culturing at 28 deg.C for 20-25h, performing first turnover 15-16h after solid culture, and performing second turnover 22-23h after solid culture.
Further, the mass percentage concentration of the brine in the step (3) is 18-20 wt%; the mass ratio of the finished koji to the saline water is 1: 2.2-2.5.
Further, the time of the first natural fermentation treatment is 15-30 d.
Further, the addition amount of the salt-tolerant rhodotorula mucilaginosa bacterial liquid in the step (3) is 2 × 105-7cfu/g soy sauce mash; the time of the second natural fermentation treatment is 50-90 days.
The soy sauce fermented by the salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield can be obtained by taking a proper amount of fermentation liquor (soy sauce) and putting the fermentation liquor into a headspace sample injection bottle, adjusting the fermentation liquor to a proper salt concentration, selecting an extraction head to finish fragrance extraction work, and determining the flavor substances by utilizing a headspace solid-phase microextraction method.
The soy sauce fermented by utilizing the salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield can form a sensory evaluation group by sensory evaluators, and the sensory evaluation group performs smell evaluation on soy sauce products and scores the taste of the flavor.
According to the invention, the salt-tolerant aroma-enhancing rhodotorula mucilaginosa is screened from the soy sauce mash, and the research on the influence of the salt-tolerant aroma-enhancing rhodotorula mucilaginosa on the flavor components of the high-salt dilute soy sauce has certain innovation. And the method is applied to the soy sauce brewing production process, and has wide potential and value for improving the flavor of the soy sauce and improving the quality of the soy sauce.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the salt-tolerant Rhodotorula mucilaginosa (Rhodotorula mucor) strain capable of highly producing phenethyl alcohol can be applied to soy sauce fermentation, the physicochemical indexes of amino nitrogen content, reducing sugar content and the like of a fermented soy sauce product are slightly improved, the relative content of volatile flavor components such as phenethyl alcohol, maltol, 2, 6-dimethyl pyrazine, furanone and the like is obviously increased, and the flavor of the soy sauce is more harmonious and lasting.
Drawings
FIG. 1 is a plate morphology of a salt-tolerant Rhodotorula mucilaginosa (Rhodotorula mucoginosa) strain with high phenylethyl alcohol yield;
FIG. 2 is a microscope image of a salt-tolerant Rhodotorula mucilaginosa (Rhodotorula mucoginosa) strain with high phenylethyl alcohol yield;
FIG. 3 is a graph showing the growth of a Rhodotorula mucilaginosa (Rhodotorula mucoginosa) strain with high phenylethyl alcohol yield;
FIG. 4 is a GC-MS total ion flow chromatogram of salt-tolerant Rhodotorula mucilaginosa (Rhodotorula mucor) strain fermented soy sauce with high phenylethyl alcohol yield.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
1. Isolation and screening of strains
Under aseptic condition, 2.5g of the mashed high-salt dilute soy sauce mash sample is taken, and is uniformly mixed with 25mL of aseptic normal saline in an aseptic sealed bag, 0.1mL of soy sauce mash sample diluent is taken to be put in a PDA flat plate, and is uniformly coated with aseptic glass beads and is cultured in an incubator at 28 ℃.
2. Identification of strains
2.1 morphological characteristics of the Strain
The colony is round, 1mm in diameter, orange, moist, smooth and raised in surface, and the plate morphology is shown in FIG. 1. The cell morphology under a microscope is shown in FIG. 2.
2.2 physiological characteristics of the Strain
The strain can grow when the NaCl concentration is 0-20%; the optimum growth pH is 5; the optimal growth temperature is 35 ℃; the strain reached logarithmic growth phase at 70h, and the growth curve is shown in FIG. 3.
2.3 molecular biological identification
Through extracting and purifying yeast genome DNA, qualified chromosome DNA is taken as a template, ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS 4 (5'-TCCTCCGCTTATTGATATGC-3') are selected as primers, 18s rRNA gene sequence PCR amplification is carried out, and gene sequence determination is completed by Beijing Liuhe Hua Dagen science and technology Limited company. The phylogenetic tree of the Rhodotorula mucilaginosa is obtained by utilizing MEGA _ X _10.0.5 software according to the result of the 18S rDNA gene sequence similarity analysis, the strain is proved to be the Rhodotorula mucilaginosa, and the strain screened by the invention is named as the salt-tolerant Rhodotorula mucilaginosa strain (Rhodotorula mucilaginosa) with high phenylethyl alcohol yield. The salt-tolerant Rhodotorula mucilaginosa strain for high yield of phenethyl alcohol is named Rhodotorula mucina, the preservation number is CGMCC No.19008, the strain is preserved in China general microbiological culture Collection center on 11 months and 26 days in 2019, and the address is microbial research institute of Zhongkoyao institute No. 3 Hospital, West Lu No.1 Hospital, the Naja district, Beijing City.
The 18S rDNA sequence of the salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield is shown as SEQ ID NO. 1.
Example 1
A method for producing fragrance of soy sauce by fermenting salt-tolerant rhodotorula mucilaginosa strains with high phenylethyl alcohol yield comprises the following steps:
(1) preparing salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield: inoculating salt-tolerant rhodotorula mucilaginosa strains with high yield of phenethyl alcohol on a malt wort liquid culture medium for activated culture, and repeating the operation for three times; centrifuging the cultured bacterial liquid at 20 deg.C and 3000r/min for 10min, collecting precipitate, washing with sterile normal saline twice, suspending in sterile normal saline to obtain bacterial liquid, and adjusting the concentration of bacterial liquid to 105CFU/mL, storing the bacterial liquid in a refrigerator at 4 ℃ and using the bacterial liquid within 24 hours;
(2) preparing high-salt dilute soy sauce: soaking soybean in water for 6h, taking out, placing the soaked soybean in an autoclave, sterilizing at 121 ℃ for 15min, uniformly mixing the cooled cooked soybean with flour and yeast extract (selected from aspergillus oryzae), wherein the mass ratio of the soybean to the flour is 4:1 according to dry weight, and the addition amount of the yeast extract is 0.05 wt% of the sum of the weight of the flour and the weight of the soybean, so as to obtain the Daqu. And putting the Daqu in an incubator for solid-state culture for 40h, setting the temperature of the Daqu to be 30 ℃ in the first 20h and setting the temperature of the Daqu to be 28 ℃ in the second 20h, and turning over the Daqu at the 15 th and 22 th hours after the solid-state culture is started to obtain finished Daqu. Mixing the finished koji with saline water with the mass percentage concentration of 18 wt%, wherein the mass of the saline water is 2.5 times that of the finished koji to obtain soy sauce mash, and naturally fermenting for 90 days;
(3) adding salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol, namely adding salt-tolerant rhodotorula mucilaginosa bacterial liquid with high yield of phenethyl alcohol obtained in the step (1) into the soy sauce mash after the natural fermentation for 20 days in the step (2) under the aseptic condition in the natural fermentation process in the step (2), wherein the adding amount is 2 × 107cfu/g of soy sauce mash, continuing fermenting for 50d, filtering and sterilizing to obtain a soy sauce sample;
(4) determination of flavor substances: and (4) putting 8mL of the soy sauce sample obtained in the step (3) into a headspace sample injection bottle, adding 0.72g of NaCl, and selecting an extraction head CAR/PDMS to finish the fragrance extraction work.
(5) Sensory evaluation: sensory evaluation groups were composed of 8 sensory evaluators (male 3, female 5), and the soy sauce products obtained in step (4) were subjected to olfactory evaluation by a sensory evaluation method in von clouds literature (research on the change rule and formation mechanism of key aroma substances of von clouds, high-salt dilute soy sauce [ D ]. university of south china, 2015.).
Comparative example 1: comparative example 1 was substantially the same as example 1 except that the halotolerant rhodotorula mucilaginosa strain was not added, and the remaining conditions were the same, to give blank sample 1.
Compared with the comparative example 1, the soy sauce flavor of the embodiment 1 is obviously improved, and the relative content of the phenethyl alcohol substance in the volatile components is improved from 0.34 to 4.32 which is 12.71 times that of the comparative example 1; the contents of 2, 6-dimethyl pyrazine, maltol and furanone substances are respectively increased by 1.80 times, 2.91 times and 7.11-8.95 times, the flavor of the soy sauce sample is more harmonious and lasting, and the flavors of sauce fragrance, mellow fragrance and the like are obviously improved.
Example 2
A method for producing fragrance of soy sauce by fermenting salt-tolerant rhodotorula mucilaginosa strains with high phenylethyl alcohol yield comprises the following steps:
(1) preparing salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield: the salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol is inoculated on a YPD liquid culture medium for activation culture, and the operation is repeated for three times. Centrifuging the cultured strain liquid at 20 deg.C and 3000r/min for 10min, collecting precipitate, washing with sterile physiological saline twice, and suspending in sterile physiological saline. Finally, adjusting the concentration of the bacterial liquid to 106CFU/mL, the bacterial suspension was stored in a 4 ℃ refrigerator and used over 24 h.
(2) Preparing high-salt dilute soy sauce: soaking soybean in water for 7h, taking out, placing the soaked soybean in an autoclave, sterilizing at 121 ℃ for 13min, uniformly mixing the cooled cooked soybean with a certain proportion of flour and yeast extract (aspergillus oryzae is selected), wherein the mass ratio of the soybean to the flour is 4:1 according to dry weight, and the addition amount of the yeast extract is 0.2 wt% of the sum of the weight of the flour and the weight of the soybean, so that the Daqu is obtained. And putting the Daqu in an incubator for solid culture for 45h, setting the temperature of the Daqu to be 30 ℃ in the first 22h and setting the temperature of the Daqu to be 28 ℃ in the second 23h, and turning over the Daqu at the 16 th and 22 th hours after the solid culture starts to obtain finished Daqu. Mixing the finished koji with 19% saline water 2.2 times of the finished koji to obtain soy sauce mash, and naturally fermenting for 90 days.
(3) Adding salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol, namely adding salt-tolerant rhodotorula mucilaginosa bacterial liquid with high yield of phenethyl alcohol obtained in the step (1) into the soy sauce mash fermented for 15 days in the step (2) under the aseptic condition in the natural fermentation process in the step (2), wherein the adding amount is 2 × 106cfu/g of soy sauce mash, continuing fermenting for 90 days, filtering and sterilizing to obtain a soy sauce sample;
(4) determination of flavor substances: and (4) putting 8mL of the soy sauce sample obtained in the step (3) into a headspace sample injection bottle, adding 0.80g of NaCl, and selecting an extraction head CAR/PDMS to finish the fragrance extraction work.
(5) Sensory evaluation: sensory evaluation groups were composed of 8 sensory evaluators (male 3, female 5), and the soy sauce products obtained in step (4) were subjected to olfactory evaluation by a sensory evaluation method in von clouds literature (research on the change rule and formation mechanism of key aroma substances of von clouds, high-salt dilute soy sauce [ D ]. university of south china, 2015.).
Comparative example 2: comparative example 2 was substantially the same as example 2 except that the halotolerant rhodotorula mucilaginosa strain was not added, and the remaining conditions were the same, to give blank sample 2.
Compared with the comparative example 2, the example 2 has the effect of obviously improving the flavor of the soy sauce, and the GC-MS total ion current chromatogram is shown in figure 4. In volatile components, the relative content of phenethyl alcohol substances is increased from 0.34 to 3.15, which is 9.26 times of that of a blank sample 2, and the contents of 2, 6-dimethyl pyrazine, maltol and furanone compounds are respectively 1.75 times, 2.25 times and 4.36-5.32 times of that of the blank sample 2, so that the flavor of a soy sauce sample is more harmonious and lasting, and the flavors of sauce, mellow and the like are obviously improved.
Example 3
A method for producing fragrance of soy sauce by fermenting salt-tolerant rhodotorula mucilaginosa strains with high phenylethyl alcohol yield comprises the following steps:
(1) preparing salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield: the salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol is inoculated on a malt wort liquid culture medium for activated culture, and the operation is repeated for three times. Centrifuging the cultured strain liquid at 20 deg.C and 3000r/min for 10min, collecting precipitate, washing with sterile physiological saline twice, and suspending in sterile physiological saline. Finally, adjusting the concentration of the bacterial liquid to 106CFU/mL, the bacterial suspension was stored in a 4 ℃ refrigerator and used over 24 h.
(2) Preparing high-salt dilute soy sauce: soaking soybean in water for 8h, taking out, placing the soaked soybean in an autoclave, sterilizing at 121 ℃ for 14min, uniformly mixing the cooled cooked soybean with a certain proportion of flour and yeast extract (aspergillus oryzae is selected), wherein the mass ratio of the soybean to the flour is 4:1 according to dry weight, and the addition amount of the yeast extract is 0.15 wt% of the sum of the weight of the flour and the weight of the soybean, so that the Daqu is obtained. And putting the Daqu in an incubator for solid-state culture for 50h, setting the temperature of the first 25h to be 30 ℃, setting the temperature of the second 25h to be 28 ℃, and simultaneously turning over the Daqu at 16h and 23h after the solid-state culture starts to obtain finished Daqu. Mixing the finished koji with saline water with the mass percentage concentration of 20 wt%, wherein the mass of the saline water is 2.3 times that of the finished koji to obtain soy sauce mash, and naturally fermenting for 90 days.
(3) Adding salt-tolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol, namely adding salt-tolerant rhodotorula mucilaginosa bacterial liquid with high yield of phenethyl alcohol obtained in the step (1) into the soy sauce mash fermented for 30 days in the step (2) under the aseptic condition in the natural fermentation process in the step (2), wherein the adding amount is 2 × 105cfu/g of soy sauce mash, continuously fermenting for 75d, filtering and sterilizing to obtain a soy sauce sample;
(4) determination of flavor substances: and (4) putting 8mL of the soy sauce sample obtained in the step (3) into a headspace sample injection bottle, adding 1.0g of NaCl, and selecting an extraction head CAR/PDMS to finish fragrance extraction.
(5) Sensory evaluation: sensory evaluation groups were composed of 8 sensory evaluators (male 3, female 5), and the soy sauce products obtained in step (4) were subjected to olfactory evaluation by a sensory evaluation method in von clouds literature (research on the change rule and formation mechanism of key aroma substances of von clouds, high-salt dilute soy sauce [ D ]. university of south china, 2015.).
Comparative example 3: comparative example 3 was substantially the same as example 3 except that the halotolerant rhodotorula mucilaginosa strain was not added, and the remaining conditions were the same, to give blank sample 3.
Compared with the comparative example 3, the soy sauce of example 3 has the effect of obviously improving the flavor of the soy sauce, the relative content of the phenethyl alcohol substances in the volatile components of the soy sauce is increased to 4.46 which is 10.37 times of that of the blank sample 1, and the contents of the 2, 6-dimethyl pyrazine, the maltol and the furanone substances are respectively 1.62 times, 1.90 times and 3.22-4.09 times of that of the blank sample 3, so that the flavor of the soy sauce sample is more harmonious and lasting, and the flavors of the sauce, the mellow flavor and the like are obviously improved. Therefore, the metabolism of the salt-tolerant rhodotorula mucilaginosa is beneficial to generating part of important flavor components in the soy sauce.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Sequence listing
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GUANGZHOU INSTITUTE OF MODERN INDUSTRIAL TECHNOLOGY
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<213> salt tolerant Rhodotorula mucilaginosa Strain (Rhodotorula mucolignosa)
<400>1
tgcctgcgga aggatcatta gtgaatatag gacgtccaac ttaacttgga gtccgaactc 60
tcactttcta accctgtgca cttgtttggg atagtaactc tcgcaagaga gcgaactcct 120
attcacttat aaacacaaag tctatgaatg tattaaattt tataacaaaa taaaactttc 180
aacaacggat ctcttggctc tcgcatcgat gaagaacgca gcgaaatgcg ataagtaatg 240
tgaattgcag aattcagtga atcatcgaat ctttgaacgc accttgcgct ccatggtatt 300
ccgtggagca tgcctgtttg agtgtcatga atacttcaac cctcctcttt cttaatgatt 360
gaagaggtgt ttggtttctg agcgctgctg gcctttacgg tctagctcgt tcgtaatgca 420
ttagcatccg caatcgaact tcggattgac ttggcgtaat agactattcg ctgaggaatt 480
ctagtcttcg gattagagcc gggttgggtt aaaggaagct tctaatcaga atgtctacat 540
tttaagatta gatctcaaat caggtaggac tacccgctga acttaagcat atcaaaagcc 600
cggaggaa 608
Claims (10)
1. A salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield is characterized in that the strain is named Rhodotorula micilaginosa with the preservation number of CGMCC No.19008, is preserved in the general microbiological center of China Committee for culture Collection of microorganisms on 11-month and 26-month 2019, and is addressed to the institute of microbiology of Zhongkoyao 3, West Siro-1 institute of North Cheng-Yang district, Beijing City.
2. The use of the halotolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol according to claim 1 in soy sauce fermentation.
3. The application of the halotolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol in soy sauce fermentation according to claim 2 is characterized by comprising the following steps:
(1) inoculating the salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield to a culture medium for activation culture to obtain a cultured bacterial liquid, centrifuging to obtain a precipitate, washing with sterile normal saline, and suspending in the sterile normal saline to obtain the salt-tolerant rhodotorula mucilaginosa bacterial liquid;
(2) adding soybeans into water, soaking, taking out to obtain soaked soybeans, carrying out high-pressure sterilization treatment on the soaked soybeans, cooling to room temperature to obtain cooked soybeans, and uniformly mixing the cooked soybeans, flour and yeast essence to obtain Daqu;
(3) carrying out solid culture on the yeast in the step (2) to obtain finished yeast; and (2) uniformly mixing the finished koji with saline water to obtain sauce mash, performing primary natural fermentation treatment on the sauce mash, then adding the salt-tolerant rhodotorula mucilaginosa bacterial liquid obtained in the step (1), performing secondary natural fermentation treatment, sterilizing, filtering and taking filtrate to obtain the soy sauce.
4. The use of the halotolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield in soy sauce fermentation according to claim 3, wherein the culture medium in step (1) is one of a wort liquid culture medium and a YPD liquid culture medium; the concentration of the salt-tolerant rhodotorula mucilaginosa bacterial liquid is 105-106CFU/mL。
5. The application of the halotolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol in soy sauce fermentation according to claim 3, wherein the soaking treatment time in the step (2) is 6-8 h; the temperature of the high-pressure sterilization treatment is 121-125 ℃, and the time of the high-pressure sterilization treatment is 10-15 min.
6. The application of the halotolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield in fermented soy sauce according to claim 3 is characterized in that the mass ratio of the soybeans to the flour in the step (2) is 4:1 in terms of dry weight; the yeast extract is Aspergillus oryzae; according to dry weight, the quality of the yeast extract is 0.05-2% of the quality of the flour and the soybean.
7. The application of the halotolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol in soy sauce fermentation according to claim 3, wherein the total time of the solid culture in the step (3) is 44-46 h; the solid state culture comprises: culturing at 30 deg.C for 22-23h, culturing at 28 deg.C for 22-24h, performing first turnover 15-16h after solid culture, and performing second turnover 22-23h after solid culture.
8. The application of the halotolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol in soy sauce fermentation according to claim 3, wherein the mass percentage concentration of the saline water in the step (3) is 18-20 wt%; the mass ratio of the finished koji to the saline water is 1: 2.2-2.5.
9. The application of the halotolerant rhodotorula mucilaginosa strain with high yield of phenethyl alcohol in soy sauce fermentation according to claim 3, wherein the time of the first natural fermentation treatment is 15-30 d.
10. The application of the salt-tolerant rhodotorula mucilaginosa strain with high phenylethyl alcohol yield in soy sauce fermentation according to claim 3, wherein the salt-tolerant rhodotorula mucilaginosa strain in the step (3) is added in an amount of2×105-7cfu/g soy sauce mash; the time of the second natural fermentation treatment is 50-90 days.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322193A (en) * | 2021-05-18 | 2021-08-31 | 华南理工大学 | Salt-tolerant yeast for increasing content of Sotolone in soy sauce and application thereof |
| CN113462585A (en) * | 2021-05-28 | 2021-10-01 | 华南理工大学 | Salt-tolerant yeast for increasing content of ethyl ester compounds in soy sauce and application thereof |
| CN113755399A (en) * | 2021-10-11 | 2021-12-07 | 上海应用技术大学 | Method for screening strains with fragrance producing and mutton smell inhibiting functional characteristics and application of method |
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| CN104178432A (en) * | 2014-08-08 | 2014-12-03 | 成都医学院 | Rhodotorula mucilaginosa and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322193A (en) * | 2021-05-18 | 2021-08-31 | 华南理工大学 | Salt-tolerant yeast for increasing content of Sotolone in soy sauce and application thereof |
| CN113322193B (en) * | 2021-05-18 | 2022-07-12 | 华南理工大学 | Salt-tolerant yeast for increasing Sotolone content in soy sauce and application thereof |
| CN113462585A (en) * | 2021-05-28 | 2021-10-01 | 华南理工大学 | Salt-tolerant yeast for increasing content of ethyl ester compounds in soy sauce and application thereof |
| CN113462585B (en) * | 2021-05-28 | 2023-02-21 | 华南理工大学 | Salt-tolerant yeast for increasing content of ethyl ester compounds in soy sauce and application thereof |
| CN113755399A (en) * | 2021-10-11 | 2021-12-07 | 上海应用技术大学 | Method for screening strains with fragrance producing and mutton smell inhibiting functional characteristics and application of method |
| CN113755399B (en) * | 2021-10-11 | 2023-01-17 | 上海应用技术大学 | Method for screening strains with fragrance producing and mutton smell inhibiting functional characteristics and application of method |
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