CN103589651A - High-salt-tolerance Zygosaccharomyces rouxii A - Google Patents
High-salt-tolerance Zygosaccharomyces rouxii A Download PDFInfo
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- CN103589651A CN103589651A CN201310362802.2A CN201310362802A CN103589651A CN 103589651 A CN103589651 A CN 103589651A CN 201310362802 A CN201310362802 A CN 201310362802A CN 103589651 A CN103589651 A CN 103589651A
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Abstract
The invention discloses a high-salt-tolerance Zygosaccharomyces rouxii A strain of which the collection number is CCTCC NO:M2013310. The strain has high salt tolerance and favorable growth stability, has the characteristic of being capable of producing full-bodied mellow and ester-like aromatic flavors, and can be used for brewing high-salt diluted soy sauce, shortening the fermentation period and improving the flavor and quality of the soy sauce.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of high salt tolerant Lu Shi zygosaccharomyces (
zygosaccharomyces rouxii).This bacterial strain can be under soy sauce brewing hypersaline environment growth and breeding, can produce the flavor of soy sauce mellow, ester is fragrant, improve soy sauce quality.
Background technology
Soy sauce is liking of a kind of traditional fermented seasonings ，Shen Shou China of China and the people of the world.Soy sauce is the product of the microbiological comprehensive effects such as aspergillus, yeast and bacterium, during fermentation, the enzyme of these microorganisms and meta-bolites form the color, smell and taste composition of soy sauce, wherein the most important thing is yeast and milk-acid bacteria, their Main Function is that fermenting carbohydrate produces the flavour substancess such as small molecular alcohol, aldehyde, acid, ester, phenols, and this is the main path that flavor of soy sauce produces.
In sauce unstrained spirits, isolated yeast has 7 genus, 32 kinds, wherein with quality of sauce relation the closest be Lu Shi zygosaccharomyces (
zygosaccharmyces rouxii), variable torulopsis (
torulopsis versatilis), cover strange torulopsis (
torulopsis mogii), changeable candiyeast (
candida versatilis) etc.Lu Shi yeast is modal osmophilic strain yeast, be characterized in being grown in the high material of sugar degree, also can be containing Fast-propagation in the culture medium of 18% salt, Main Function is to carry out zymamsis, meanwhile carries out synthetic etc. as synthetic, the glycerine of ester class and polyvalent alcohol of various aroma components.
Improved in high-salt dilute zymotechnique Shi China traditional zymotic technique, assimilated again Japanese many modern techniquies simultaneously, fermentation period is 3-6 month, and soy sauce product amino-acid nitrogen production rate is high, is about 60% left and right of full nitrogen, and flavor of soy sauce is good.In high salt liquid soy mash fermenting process, in order to improve the local flavor of soy sauce, generally all to add salt tolerant aroma-producing yeast, thereby improve the single and nonsaturation of aspergillus oryzae fermented flavour for soy sauce brewing, improve local flavor and the class of soy sauce comprehensively.The adding technique of Lu Shi yeast is ripe, applies more extensively, is taste effect and gains public acceptance.
Yet the artificial yeast adding may be not suitable with the hypersaline environment in soy sauce brewing process, can not play a role well.Therefore screening halophilic saccharomyces soya bacterial strain is one of important means of improving flavor of soy sauce, raising quality of sauce.
Summary of the invention
In view of the deficiencies in the prior art, the object of the present invention is to provide halophilic product alcohol, the ester-producing yeast bacterial strain that is applied to soy sauce brewing of a strain.This bacterial strain can be under soy sauce brewing hypersaline environment growth and breeding, can produce the flavor of soy sauce mellow, ester is fragrant, improve soy sauce quality.According to its morphological specificity, physiological and biochemical property and the result for retrieval of 26S rRNA gene order in Genbank thereof, identify strains A be Lu Shi zygosaccharomyces (
zygosaccharmyces rouxii).
The object of the present invention is achieved like this:
A kind of high salt tolerant Lu Shi zygosaccharomyces A(
zygosaccharomyces rouxii A) bacterial strain, on 07 02nd, 2013, being preserved in Chinese Typical Representative culture collection center, preserving number is CCTCC NO:M2013310.
The 26S rRNA nucleotide sequence of above-mentioned high salt tolerant Lu Shi zygosaccharomyces A bacterial strain is as shown in SEQ ID NO:1.
High salt tolerant Lu Shi zygosaccharomyces A(provided by the present invention
zygosaccharomyces rouxii A) bacterial strain has following morphological specificity: diameter 5 μ m, spherical, do not move, in the mode of sprouting, carry out vegetative propagation.On YEPD nutrient agar, cultivate after 48h for 28 ℃, be oyster white, opaque, circular bacterium colony.
High salt tolerant Lu Shi zygosaccharomyces A(provided by the present invention
zygosaccharomyces rouxii A) whole-cell fatty acid of bacterial strain mainly exists with oleic acid, palmitinic acid, Zoomeric acid and stearic acid form.Containing on salt culture medium, cultivating, strains A unsaturated fatty acid content increases, and its whole-cell fatty acid mainly exists with oleic acid, linolic acid, Zoomeric acid and palmitinic acid form.
According to salt resistant character experimental result, high salt tolerant Lu Shi zygosaccharomyces A(provided by the present invention
zygosaccharomyces rouxii A) the salt tolerant scope (take NaCl) of bacterial strain is 0-24%.
According to temperature growth experiment result, high salt tolerant Lu Shi zygosaccharomyces A(provided by the present invention
zygosaccharomyces rouxii A) growth temperature range of bacterial strain is: 6-39 ℃.
High salt tolerant Lu Shi zygosaccharomyces A(of the present invention
zygosaccharomyces rouxii A) bacterial strain cultivates mellow, the ester Flavor that produce and be obviously better than YEPD substratum in the YEPD of 20%NaCl substratum, for soy sauce brewing, can improve flavor of soy sauce, improves soy sauce quality.
High salt tolerant Lu Shi zygosaccharomyces A(of the present invention
zygosaccharomyces rouxii A) bacterial strain is at 28 ℃ of cultivation 24h of YEPD substratum, culture is counted in continuous passage, and its cultural characteristic and morphological specificity have no significant change, and biological character is stable.
Compared with prior art, high salt tolerant Lu Shi zygosaccharomyces A(of the present invention
zygosaccharomyces rouxii A) bacterial strain has following useful technique effect:
(1) according to the salt tolerance experiment of embodiment 3 tables 2, can find out, Lu Shi zygosaccharomyces bacterial strain of the present invention exists in situation growing state good at 20% sodium-chlor, at 24% sodium-chlor, exist in situation and still can keep certain increment, at 26% sodium-chlor, exist in situation and still can grow.This illustrates that Lu Shi zygosaccharomyces bacterial strain salt tolerance of the present invention is strong, is applicable to the application in industrial high-salt liquid-state brewing soy sauce production completely.
(2) Lu Shi zygosaccharomyces bacterial strain of the present invention growing state at 35 ℃ is good, at 39 ℃, still can keep certain increment.This illustrates that Lu Shi zygosaccharomyces bacterial strain of the present invention has certain temperature tolerance, is applicable to industrial application in high-salt liquid-state brewing soy sauce is produced.
(3), according to embodiment 4, Lu Shi zygosaccharomyces bacterial strain of the present invention is cultivated after 4d 28 ℃ of the YEPD substratum containing 18% sodium-chlor, produces aroma substance, has stronger mellow, ester Flavor.
(4) Lu Shi zygosaccharomyces bacterial strain of the present invention, because salt tolerance is high, can shorten fermentation period in high-salt dilute soy is brewageed, and produces the flavor of soy sauce mellow, ester is fragrant, for the industrial production of soy sauce provides strain excellent.
Accompanying drawing explanation
Fig. 1 is that Lu Shi zygosaccharomyces A of the present invention is at 1000 power microscope thalli morphology figure.
Fig. 2 is that Lu Shi zygosaccharomyces A of the present invention is based on 26S rRNA gene development tree graph.
Embodiment
Below in conjunction with embodiment, describe the present invention.Embodiment is only for illustrating, and scope of the present invention is not limited with specific implementation method, but is limited by the scope of claim.
Embodiment 1: the 26S rRNA identification experiment of Lu Shi zygosaccharomyces A of the present invention
The extraction of pastoris genomic dna and purifying utilize Sambrook et al. (1989) method.Take and extract and detect qualified chromosomal DNA as template, select NL 1 (5'-GCATATCAATAAGCGGAGGAAAAG-3 ') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3 ') as primer, the pcr amplification of 26s rRNA gene order is pressed Solieri L. et al.(2007) method operation, gene sequencing is completed by Shanghai Ying Jun company.Utilize Clustalx1.8, MEGA 5.0 softwares (Tamura et al. 2011), according to the result of 26S rRNA gene order similarity analysis, choose the 26S rRNA gene order of relevant bacterial strain, adopt respectively neigbor-joining(ortho position phase connection, NJ) (Saitou and Nei 1987) algorithm obtains corresponding phylogenetic tree (Fig. 2).The phylogenetic tree building shows that strains A exists
zygosaccharomycesbelong on branch of evolutionary tree, with
z. Rouxiikind bacterial strain in a monoid and from other kinds in different branches.Illustrate strains A belong to Lu Shi zygosaccharomyces (
zygosaccharmyces rouxii).
Embodiment 2: Lu Shi zygosaccharomyces A whole-cell fatty acid component characteristics analysis experiment of the present invention
During yeast growth, be to rely on the saturation ratio of its cell membrane fat acid to adapt to the unfavorable factors such as alcohol in surrounding environment, salt, high temperature.In order to analyze the fatty acid component of strains A, first strains A is inoculated in YEPD meat soup, 28 ℃ are cultured to the logarithmic phase later stage, collect suitable thalline, the method of describing according to < < the protocol of the Sherlock Microbial Identification System (MIDI) > > (version 6.0.) is extracted thalline fatty acid methyl ester, finally by Agilent 6890N GC gas-chromatography, pass through MIDI Sherlock YEAST6 database analysis fatty acid component (Sasser, 1990).Strains A whole-cell fatty acid component of the present invention is as shown in table 1.Strains A whole-cell fatty acid mainly exists with oleic acid, palmitinic acid, Zoomeric acid and stearic acid form.On saliferous (18% NaCl) substratum, cultivate, strains A unsaturated fatty acid content increases, and C18 fatty acid content increases, and its whole-cell fatty acid mainly exists with oleic acid, linolic acid, Zoomeric acid and palmitinic acid form.Cultivate and compare on its high sodium chloride concentration YEPD substratum with existing Lu Shi zygosaccharomyces, the C18 fatty acid content of strains A has improved more than 5%, and unsaturated fatty acid content has improved more than 10%.
Table 1 bacterium A main fatty acid composition
Embodiment 3: the salt tolerant experiment of Lu Shi zygosaccharomyces A of the present invention
Preparation substratum (1% yeast powder, 5% glucose, 2% peptone, KH
2pO
40.1%, KCl 0.04%, CaCl
20.11%, (NH
4)
2sO
40.5%, MnSO
40.25 mg%, FeCl
30.25 mg%), add NaCl, setting salt concn is 16%, 18%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, accesses strains A of the present invention after moist heat sterilization, put 28 ℃, 200 revs/min cultivation observation substratum and become the muddy time, result is as shown in table 2.Strains A of the present invention exists in situation growing state good at 20% sodium-chlor, at 24% sodium-chlor, exists in situation and still can keep certain increment, at 26% sodium-chlor, exists in situation and still can grow.And existing Lu Shi zygosaccharomyces generally exists in situation growing state better being less than 16% sodium-chlor, at 20% sodium-chlor, exists in situation and almost can not grow.
Table 2 strains A salt tolerant experiment of the present invention
Embodiment 4: Lu Shi zygosaccharomyces A of the present invention is testing containing producing perfume in the YEPD substratum of 18% sodium-chlor
With transfering loop, from test tube slant, get Lu Shi zygosaccharomyces bacterial strain of the present invention one ring and be inoculated in the 250 mL triangular flasks that fill 30 mL liquid nutrient mediums, in 28 ℃, 180 r/min shake-flask culture 10 h, the light absorption value of seed liquor reaches 2.4 left and right.Under aseptic technique, draw activation solution 2 mL and be inoculated in 200 mL containing in the YEPD liquid nutrient medium of 18% sodium-chlor, set 28 ℃ of leavening temperatures, rotating speed 180/min, cultivates after 4d, has stronger mellow, ester Flavor.Adopt headspace sampling, utilize GC-MS to measure aroma component in substratum.Analysis condition is: solid-phase microextraction condition: get 15g fermented liquid and be put in 50 mL head space bottles, 60 ℃ are placed in magnetic stirring apparatus water-bath balance 15min.Then use DVB/ CAR/ PDMS 50/ 30 μ m (Vinylstyrene/carbonaceous molecular sieve/polydimethylsiloxane) extracting head head spaces to adsorb after 40 min, extracting head is inserted to GC sample introduction, resolve 5 min.Chromatographic condition: column type adopts nonpolar capillary column 60 μ m * 0.25, the m * 250 μ m of Agilent HP-5ms; Temperature programming: 40 ℃ of the initial temperature speed with 2.5 ℃/min is warmed up to 130 ℃, keeps 1min.Speed with 8 ℃/min is warmed up to 250 ℃ of maintenance 1 min again; 250 ℃ of injector temperatures, do not shunt; Carrier gas is helium; Volumetric flow rate is 1.0mL/ min.Mass spectrum condition: ionization mode is EI; Electron energy 70 eV; Voltage 350V; 280 ℃ of Link Port temperature; 230 ℃ of ion source temperatures; 150 ℃ of quadrupole temperature; Quality of scanning: 32 ~ 450amu/sec.Key odorant composition is as shown in table 3.Compare with existing Lu Shi zygosaccharomyces, strains A can improve more than 15% containing cultivating the total alcohol output producing in high density chlorination sodium YEPD liquid nutrient medium, and total ester content can improve more than 10%.
Table 3 strains A of the present invention is at the YEPD culture medium culturing key odorant composition containing 18% sodium-chlor
Sequence table
SEQUENCE LISTING
<110> Hubei University Of Technology
Mono-kind high salt tolerant Lu Shi zygosaccharomyces of <120>
<160> 1
<210> 1
<211> 582
<212> DNA
The 26S rRNA nucleotide sequence of <213> Lu Shi zygosaccharomyces A bacterial strain
<400> 1
aaaccaaccg ggattgcctt agtaacggcg agtgaagcgg caaaagctca aatttgaaat 60
ctggtacctt tcggtgcccg agttgtaatt tggagaaagt gattctggga ctggcccttg 120
cctatgttcc ttggaacagg acgtcataga gggtgagaac cccgtgaggc gaggtgatcc 180
agttctttgt agaacgcttt cgaagagtcg agttgtttgg gaatgcagct ctaagtgggt 240
ggtaaattcc atctaaagct aaatacaggc gagagaccga tagcgaacaa gtacagtgat 300
ggaaagatga aaagaacttt gaaaagagag tgaaaaagga cgtgaaattg ttgaaaggga 360
agggcatttg atcagacatg gtgttttgtg cccctcgctc ctcgtgggtg ggggaatctc 420
gcagctcact gggccagcat cagttttggc ggcaggataa atctctggga atgtggcttc 480
tttcttcggg agggagtgtt atagcccagg ggaatactgc cagctgggac tgaggtatgc 540
gacattttgt caaggatgtt ggcataatgg ttatatgccg cc 582
Claims (10)
1. one kind high salt tolerant Lu Shi zygosaccharomyces A(
zygosaccharomyces rouxii A) bacterial strain, its deposit number is CCTCC NO:M2013310.
2. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, is characterized in that: the 26S rRNA nucleotide sequence of described bacterial strain is as shown in SEQ ID NO:1.
3. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1 and 2, it is characterized in that: the diameter 5 μ m of described bacterial strain, spherical, do not move, in the mode of sprouting, carry out vegetative propagation, be oyster white, opaque circular bacterium colony after 28 ℃ of YEPD nutrient agars are cultivated 48h.
4. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, it is characterized in that: the whole-cell fatty acid of described bacterial strain mainly exists with oleic acid, palmitinic acid, Zoomeric acid and stearic acid form, containing on salt culture medium, cultivating, full cell unsaturated fatty acid content increases, and its whole-cell fatty acid mainly exists with oleic acid, linolic acid, Zoomeric acid and palmitinic acid form.
5. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, is characterized in that: the sodium chloride tolerance concentration of described bacterial strain is 0-24%.
6. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, is characterized in that: the glucose-tolerant concentration of described bacterial strain is 0-60%.
7. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, is characterized in that: the growth temperature range of described bacterial strain is 6-39 ℃.
8. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, is characterized in that: the growth pH scope of described bacterial strain is 4.0-7.0.
9. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, is characterized in that: described bacterial strain is being cultivated containing in the YEPD substratum of salt, produces aroma substance, has stronger mellow, ester Flavor.
10. high salt tolerant Lu Shi zygosaccharomyces A bacterial strain according to claim 1, is characterized in that: described bacterial strain is cultivated 24h 30 ℃ of YEPD substratum, and culture is counted in continuous passage, and its cultural characteristic and morphological specificity have no significant change, and biological character is stable.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104886529A (en) * | 2015-06-09 | 2015-09-09 | 浙江五味和食品有限公司 | High-salt and high-alcohol diluted state soy sauce rapid brewing method |
| CN105695349A (en) * | 2016-04-15 | 2016-06-22 | 湖北工业大学 | Method using phosphorus starvation culture to increase intracellular trehalose of yeast cells |
| CN108315271A (en) * | 2018-04-09 | 2018-07-24 | 佛山市海天(高明)调味食品有限公司 | One primary yeast and its application |
| CN108902748A (en) * | 2018-08-24 | 2018-11-30 | 江南大学 | A kind of method of urethanes in reduction thick broad-bean sauce |
| CN109321479A (en) * | 2018-11-27 | 2019-02-12 | 宜宾五粮液股份有限公司 | Acidproof saccharomyces cerevisiae and application thereof |
| CN110229760A (en) * | 2019-04-30 | 2019-09-13 | 四川省食品发酵工业研究设计院 | One plant has flavouring, the dedicated Lu Shi Zygosaccharomyces of fermentation sauce for the function that loses lustre and its application |
| CN110408554A (en) * | 2019-08-23 | 2019-11-05 | 李锦记（新会）食品有限公司 | One plant of heat-resisting Zygosaccharomyces FW60-5 and its application |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104886529A (en) * | 2015-06-09 | 2015-09-09 | 浙江五味和食品有限公司 | High-salt and high-alcohol diluted state soy sauce rapid brewing method |
| CN105695349A (en) * | 2016-04-15 | 2016-06-22 | 湖北工业大学 | Method using phosphorus starvation culture to increase intracellular trehalose of yeast cells |
| CN105695349B (en) * | 2016-04-15 | 2019-04-05 | 湖北工业大学 | A kind of method of phosphate starvation culture and improvement yeast cells intracellular trehalose |
| CN108315271A (en) * | 2018-04-09 | 2018-07-24 | 佛山市海天(高明)调味食品有限公司 | One primary yeast and its application |
| CN108902748A (en) * | 2018-08-24 | 2018-11-30 | 江南大学 | A kind of method of urethanes in reduction thick broad-bean sauce |
| CN109321479A (en) * | 2018-11-27 | 2019-02-12 | 宜宾五粮液股份有限公司 | Acidproof saccharomyces cerevisiae and application thereof |
| CN110229760A (en) * | 2019-04-30 | 2019-09-13 | 四川省食品发酵工业研究设计院 | One plant has flavouring, the dedicated Lu Shi Zygosaccharomyces of fermentation sauce for the function that loses lustre and its application |
| CN110229760B (en) * | 2019-04-30 | 2022-11-01 | 四川省食品发酵工业研究设计院有限公司 | Special zygosaccharomyces rouxii for fermented sauce with functions of increasing aroma and reducing color and application thereof |
| CN110408554A (en) * | 2019-08-23 | 2019-11-05 | 李锦记（新会）食品有限公司 | One plant of heat-resisting Zygosaccharomyces FW60-5 and its application |
| CN110408554B (en) * | 2019-08-23 | 2021-04-23 | 李锦记（新会）食品有限公司 | Heat-resistant zygosaccharomyces FW60-5 and application thereof |
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