CN112852667B - Method for increasing content of flavor substances of soy sauce - Google Patents
Method for increasing content of flavor substances of soy sauce Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/50—Soya sauce
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a method for improving the content of soy sauce flavor substances, and belongs to the technical field of bioengineering. The invention screens and separates bacterial strains from soy sauce mash, screens and obtains a Weissella mesenteroides with increased soy sauce flavor substance content, inspects the influence of the commonly enhanced Weissella mesenteroides and zygosaccharomyces rouxii on the soy sauce flavor substance content by using a general high-salt dilute soy sauce fermentation system, and finds that the commonly enhanced two bacteria can obviously improve the soy sauce flavor substance content. In the process of fermenting the high-salt dilute soy sauce, compared with the control without strengthening any strain, the jointly strengthened Weissella and zygosaccharomyces rouxii can increase the generation amount of the flavor substance content in the system by 2.1 times, improve the total content of the organic acid by 72.76 percent and improve the total content of the free amino acid by 33.15 percent.
Description
Technical Field
The invention relates to a method for improving the content of flavor substances of soy sauce, belonging to the technical field of bioengineering.
Background
The main components of the soy sauce comprise free amino acids, polypeptides, organic acids and volatile flavor substances, wherein the free amino acids and the volatile flavor substances influence the taste and the flavor of the soy sauce and are key substances influencing the quality of the soy sauce. The variety and content of volatile flavor substances have important influence on the flavor of the soy sauce, and the unique flavor of the soy sauce is derived from succession of microbial communities and metabolic regulation. By summarizing the research reports related to soy sauce aroma substances, 1038 volatile compounds are identified and finished together. These compounds fall into two main categories: aromatic compounds and aliphatic compounds, further subdivided to include alcohols, acids, esters, phenols, aldehydes, ketones, furans, pyrazines, and the like.
The flavor substances of the soy sauce are improved mainly from two aspects: 1. the fermentation process is improved. The soy sauce flavor substances are influenced by fermentation factors and fermentation processes, wherein the content of the flavor substances can be effectively improved by regulating and controlling multiple factors such as fermentation temperature, fermentation time, carbon-nitrogen ratio, fermentation capacity, saccharification capacity and the like; in addition, the fermentation conditions such as temperature, pH, oxygen content, etc. of the fermentation process also have an effect. The defects of the method are that the workload is large, the time and the labor are consumed, and the efficiency is low due to excessive influence factors; 2. a microbial means. The screening of aroma-enhancing microorganisms is the most important means for improving the content of flavor substances at present, and the multi-strain fermentation can improve the diversity and activity of enzymes and can better improve the flavor substances of the soy sauce, but the strain screening process is slow, so that the quality improvement of the soy sauce is influenced. Therefore, strengthening functional microorganisms in soy sauce fermentation is currently the safest and most effective method.
The soy sauce mash is one of main environments where important functional microorganisms inhabit in the soy sauce fermentation process, free amino acids and organic acids generated by bacterial metabolism of the soy sauce mash are main precursors of main fragrances HEMF and 4-EG of soy sauce, and have important influence on the quality and flavor of the soy sauce. In the continuous acclimation and dynamic flora replacement process of the soy sauce fermentation high-salt environment, the functional microbial flora adapted to the soy sauce mash environment is gradually enriched, and the generation of flavor substances in the soy sauce fermentation process can be improved. Therefore, the strengthening of the functional microbial flora in the high-salt dilute soy sauce mash has important significance for promoting the application of improving the content of the flavor substances in the soy sauce fermentation process.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a method for improving the content of soy sauce flavor substances.
The first purpose of the invention is to provide a Weissella mesenteroides (Weissellaparameteroides) LCW-28 which is preserved in China center for type culture Collection at 11-27 months in 2020 with the preservation number of CCTCCM 2020778.
The second purpose of the invention is to provide a culture method of Weissella mesenteroides LCW-28, which is to inoculate Weissella mesenteroides LCW-28 to MRS culture medium and culture the Weissella mesenteroides LCW-28 statically at 37 ℃.
The third purpose of the invention is to provide a starter which contains Weissella mesenteroides LCW-28.
In one embodiment of the present invention, the preparation method of the leavening agent is: inoculating 200-600 mu L of Weissella mesenteroides LCW-28 into 10-30 mLMRS liquid culture medium, activating for 2-3 generations at 37 ℃, and waiting until Weissella mesenteroides LCW-28 reaches 1.0 multiplied by 107When the number of viable bacteria is more than CFU/mL, centrifuging at 6000-10000 rpm15min, removing supernatant, sequentially adding buffer solution and cryoprotectant in sterile environment until cell concentration is not less than 1.0 × 107And (5) performing vacuum freeze drying treatment at CFU/mL to obtain the leaven.
In one embodiment of the present invention, the buffer solution is 0.1-0.3M phosphate buffer solution with pH 6-7 and/or 0.5-1% physiological saline and/or double distilled water, and the cryoprotectant is 10-20% glycerol and/or 8-12% trehalose.
The fourth purpose of the invention is to provide a method for improving the content of flavor substances of soy sauce, which is to add the Weissella mesenteroides LCW-28 or the leavening agent in the soy sauce fermentation process.
In one embodiment of the invention, the Weissella mesenteroides LCW-28 or starter culture has a final concentration of 1.0X 10 in the fermentation system6～1.0×108CFU/g。
In one embodiment of the invention, the Weissella mesenteroides LCW-28 is present in a mash environment having a mash concentration of 18% of at least 1.0 x 107CFU/g。
In one embodiment of the present invention, yeast is also added during the soy sauce fermentation.
In one embodiment of the invention, the weissella mesenteroides LCW-28 or starter is added simultaneously with the yeast during soy sauce fermentation.
In one embodiment of the invention, the yeast is added to the Weissella mesenteroides LCW-28 or the leavening agent in a ratio of 1:1 during soy sauce fermentation.
In one embodiment of the present invention, the yeast is ZQ-01, Zygosaccharomyces rouxii.
The fifth purpose of the invention is to provide the application of the Weissella mesenteroides LCW-28 or the leavening agent or the method for improving the content of the flavor substances in the preparation of soy sauce.
Has the advantages that:
the Weissella mesenteroides LCW-28 strain capable of remarkably improving the generation of flavor substances in the soy sauce fermentation process is obtained by separating and purifying soy sauce mash microorganisms. The strain and the strain ZQ-01 are strengthened together, so that the content of flavor substances generated in a system can be well improved, and the content of the flavor substances can be improved by 2.1 times compared with a Control group without the added strain. Meanwhile, compared with a Control group without adding the bacterial strains, the total content of the organic acid of the two bacteria is increased by 72.76%, and the total content of the free amino acid is increased by 33.15%. The amino nitrogen of the two-bacterium reinforced group is improved by 97.4 percent compared with the Control group without the added strain on the 40 th day of fermentation. Therefore, the Weissella mesenteroides LCW-28 is a strain which is suitable for the soy sauce fermentation production environment and can obviously improve the content of flavor substances in the fermentation process, and the content of the flavor substances in the soy sauce can be well controlled within a proper range by adjusting the addition amount of the strain, so that the Weissella mesenteroides LCW-28 has important significance for improving the flavor substances in the soy sauce fermentation production.
Biological material preservation
The Weissella mesenteroides provided by the invention is classified and named as Weissellaparameteroides LCW-28, is preserved in China center for type culture Collection at 11/27 of 2020, has the preservation number of CCTCCM2020778, and has the preservation address of Wuhan university in China.
Drawings
FIG. 1 shows the dynamic changes of the amounts of Weissella mesenteroides (a) and Zygosaccharomyces rouxii (b) in the fermentation process of high-salt dilute soy sauce.
FIG. 2 shows the changes of physicochemical indexes (a is the change of reducing sugar, b is the change of pH, c is the change of total acid, and d is the change of amino nitrogen) of singly reinforced Weissella mesenteroides LCW-28 and singly reinforced zygosaccharomyces rouxii ZQ-01.
FIG. 3 shows the changes in organic acid content for the individual enrichment of Weissella mesenteroides LCW-28, the individual enrichment of ZQ-01, and the joint enrichment of both bacteria.
Detailed Description
ZQ-01: the mechanism of production of ethyl carbamate in soy sauce and elimination strategy described in sarean, study [ D ]. university of south of the river, 2016.
MRS culture medium: 10g/L of peptone, 8g/L of beef extract powder, 4g/L of yeast extract powder, 20g/L of glucose, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 801g/L of tween.
Bengal red medium: 5g/L of peptone, 10g/L of glucose, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 20g/L of agar and 100mL/L of 1/3000 Bengal solution.
YPD medium: 10g/L of yeast extract, 20g/L of peptone and 20g/L of glucose.
Example 1: screening of strains
With vancomycin (0.2 g. L)-1) And natamycin (0.1 g.L)-1) The MRS medium of (1) was isolated and screened from the moromia. Selecting sauce mash samples fermented for 5, 15 and 25 days, taking 15g of sauce mash and placing the sauce mash into a beaker, adding 135mL of sterile normal saline and adding a proper amount of glass beads, and stirring at 100 r.min-1Oscillating for 5min, standing at room temperature for 5min, taking 1mL of bacterial suspension, diluting the bacterial suspension in gradient, and coating on vancomycin (0.2 g. L)-1) And natamycin (0.1 g.L)-1) The MRS medium of (1) is subjected to inverted culture at 37 ℃ for 1-3 days. Single colonies were picked from the plates and inoculated into 50mL of a suspension containing vancomycin (0.2 g. L)-1) And natamycin (0.1 g.L)-1) The MRS liquid culture medium is kept still for 24 hours at 37 ℃ and 8000r/min, and thalli are collected by centrifugation.
Extracting the genome of the strain by using a commercial gene extraction kit, respectively amplifying the 16SrRNA gene sequence and the recN gene sequence of the strain by using a 16SrRNA general primer and a recN gene specific primer (table 1) as templates, sending the amplified products to Shanghai bioengineering company Limited for sequencing, and comparing and analyzing the sequencing result with the sequence in an NCBI database.
TABLE 1PCR primers
Example 2: soy sauce preparation using Weissella mesenteroides LCW-28 and ZQ-01
(1) Strain culture:
culturing Weissella mesenteroides LCW-28: selecting Weissella mesenteroides single colony from a microorganism activation plate, inoculating the Weissella mesenteroides single colony into a liquid MRS culture medium, standing and culturing at 37 ℃ for 24h, inoculating the Weissella mesenteroides single colony into a fresh MRS culture medium according to the proportion of 1% (v/v), and culturing until OD is obtained600To 3.0.
Culture of ZQ-01 of ZQ-Lu's yeast: respectively picking single yeast colony from microorganism activation plate, inoculating into liquid YPD medium, culturing at 37 deg.C and 220r min-1Culturing for 30h, inoculating to fresh YPD medium at a ratio of 1% (v/v), and culturing to OD600To 4.0.
(2) The fermentation process of the high-salt dilute soy sauce comprises the following steps:
a starter propagation process: soaking bean cake in 1.2 times of water for 2 hr, sterilizing at 121 deg.C for 15min, and cooling to room temperature. Mixing soy sauce koji (1.5 ‰ (w/w) of total weight of raw materials) with appropriate amount of parched wheat grains, and mixing with soybean meal and wheat grains (w/w is 6: 4). Culturing at 30 deg.C in biochemical incubator, turning over at appropriate time, making starter for 48h, and collecting yeast material with yellow-green mycelium on surface.
The fermentation process comprises the following steps: mixing the finished koji with a mixture containing 200 g.L-1NaCl brine was mixed in a volume ratio of 1: 1.8. And (3) filling the uniformly mixed sauce mash into 500mL beakers, and fermenting for 30 days at 30 ℃. The fermentation was stirred once daily for week 1, and every two days thereafter. Samples were taken on days 0, 5, 10, 15, 20, 25, 30.
The strengthening mode is as follows: the adding mode of the bacterial strain in the fermentation process of the high-salt dilute soy sauce is as follows: adding 10 at 0 day of fermentation7CFU·g-1ZQ-01 of ZQ-01 and simultaneously adding 107CFU·g-1Weissella mesenteroides LCW-28.
Five experimental groups are arranged in total, the specific implementation mode is as above,
control group: no strain is added;
rouxii group: zygosaccharomyces rouxii alone is fortified;
parametenteroides group: strengthening Weissella mesenteroides alone;
co-encapsulation group: simultaneously strengthening Weissella mesenteroides and zygosaccharomyces rouxii;
sequentialinocilation: is to inoculate Weissella mesenteroides and the like at first and then inoculate zygosaccharomyces rouxii when the pH value is reduced to 5.
Example 3: determination of quantities of Weissella mesenteroides LCW-28 and ZQ-01
In order to confirm that Weissella mesenteroides and Zygosaccharomyces rouxii can be enhanced by adding strains during soy sauce fermentation, a plate counting method is adopted for measuring the quantity of Weissella mesenteroides and yeast. Weighing 1g of fresh soy sauce mash or co-culture bacteria liquid, pouring into a conical flask containing sterilized glass beads and 99mL of physiological saline, mixing uniformly, and diluting in a gradient manner (respectively diluting to 10)-1、10-2、10-3、10-4、10-5、10-6) Diluted and spread on MRS medium and Bengal medium, and cultured at 37 ℃ for 1 day, and counted. Weissella mesenteroides is facultative anaerobe, and bacterial colony appears in plate culture for 1-2 days. The predominant salt-tolerant bacteria in the sauce mash is the halophilic tetragenococcus, and bacterial colonies appear after 4-6 days of plate culture. Thus, MRS plate counts can be distinguished from the dominant salt-tolerant bacteria in moromi (including Weissella, Bacillus, Staphylococcus). The yeast amount calculated by the method comprises the added zygosaccharomyces rouxii and salt-tolerant yeast (such as torulopsis, candida and the like) in the sauce mash. The latter are less numerous and negligible compared to the former.
The result shows that the addition of Weissella mesenteroides LCW-28 can increase the number of Weissella mesenteroides in the sauce mash by 1.9-3.0 orders of magnitude compared with the Weissella mesenteroides contrast which is not added, and the maximum value reaches 1.0 multiplied by 108CFU·g-1Gradually decreases in the later stage and stabilizes at 1.0 × 107CFU·g-1. Therefore, the addition of Weissella mesenteroides LCW-28 can increase the concentration of the Weissella mesenteroides LCW-28 in the soy sauce fermentation process and strengthen the strain (figure 1).
Example 4: the reinforced influence of Weissella mesenteroides LCW-28 and ZQ-01 on the physicochemical indexes of the soy sauce mash sample
The moromi of example 1 was sampled on days 0, 5, 10, 15, 20, 25, 30, 35 and 40. Taking 100g of sauce mash in a centrifuge tube, and carrying out 12000 r.min-1Centrifuging for 30min, collecting supernatant, and filtering with filter membrane with pore diameter of 0.22 μm for physicochemical index determination. The pH of the soy sauce mash is directly measured by a precise pH meter. The total acid content was determined by acidimetry. The content of amino acid nitrogen is measured by adopting a formaldehyde titration method. The content of reducing sugar is determined by 3, 5-dinitrosalicylic acid method (DNS).
Analysis of physicochemical indexes such as pH, total acid, reducing sugar and amino acid nitrogen of the moromi mash in the high-salt dilute soy sauce fermentation process shows that the pH of the moromi mash is slightly reduced by adding Weissella mesenteroides, the total acid content is slightly increased, the content of the amino acid nitrogen is obviously increased, as shown in figure 2, at the 40 th day, compared with a Control group, the pH of the moromi mash is reduced by 15% by strengthening two bacteria (Co-infection group), the total acid is increased by 41.28%, the reducing sugar is decreased by 132.8%, and the amino nitrogen is increased by 97.4%. The amino nitrogen is used as the main judgment standard of the national soy sauce quality, and the content of the amino nitrogen can be obviously improved by jointly strengthening the two bacteria.
Example 5: effect of enhanced Weissella mesenteroides LCW-28 and ZQ-01 on free amino acids
The free amino acid is measured by high performance liquid chromatography, and is subjected to in-column derivatization by adopting o-xylene (OPA), and the specific measurement method is referred to documents. Diluting the soy sauce filtrate by 20 times with 5% trichloroacetic acid, treating in dark for 30min, filtering with water system filter membrane with pore diameter of 0.22 μm, and collecting 1mL sample. This operation can remove the interference of the protein on the measurement result. Chromatographic conditions are as follows: chromatograph Agilent1260, column ODSYPERSIL (250 mm. times.4.6 mm. times.5 μm). The mobile phase A and the mobile phase B are prepared as before, and are filtered and subjected to ultrasonic treatment for more than 30min before use. Mobile phase a phase: 5 g.L-1Sodium acetate solution (containing 0.2 mL. L)-1 Triethylamine 5 mL. L-1Tetrahydrofuran), pH adjusted to 7.2 with acetic acid. Mobile phase B phase: 5 g.L-1Sodium acetate-methanol-acetonitrile solution with the volume ratio of 1:2: 2.
The free amino acid has important contribution to the formation of unique taste and aroma of fermented foods, particularly the fermented soybean foods taking protein as a main fermentation substrate. The content of free amino acid is one of the main physicochemical indexes for measuring the quality of soy sauce. Glutamic acid is umami amino acid and is main free amino acid in the umami taste of the soy sauce. Glycine and threonine are sweet amino acids. Compared with the Control group, the total content of free amino acid of the two reinforced bacteria (Co-inoculation group) is improved by 33.15 percent. Wherein glutamic acid is improved by 50%, lysine is improved by 56.39%, glycine is improved by 38.62%, and threonine is improved by 70.79%.
TABLE 2 Change in free amino acids of enhanced Weissella mesenteroides and Zygosaccharomyces rouxii (mg. mL)-1)
Example 6: effect of enhanced Weissella mesenteroides LCW-28 and ZQ-01 on organic acid content
The organic acid is determined by high performance liquid chromatography, and the specific method is referred to in the literature. The filtrate of the moromi mash was diluted 10 times with ultrapure water, and then filtered through a water-based filter membrane having a pore size of 0.22 μm, and 1mL of a sample was sampled and measured. Chromatographic conditions are as follows: liquid phase system: agilent 1260; a chromatographic column: an organic acid column; an ultraviolet detector: 210 nm; sample introduction volume: 10 mu L of the solution; mobile phase: 5 mmol. L-1 dilute sulfuric acid; column temperature: 40 ℃; elution speed: 0.5 mL/min-1(ii) a Separation time: and (3) 30 min.
The organic acid generated by lactic acid bacteria in the soy sauce is not only an important component of soy sauce flavor substances, but also plays a role in inhibiting the growth of putrefying bacteria or mixed bacteria in soy sauce mash, and the total content of organic acid for strengthening two bacteria (Co-inoculation group) is improved by 72.76 percent compared with the Control group at the 40 th day. In which propionic acid, which is a short chain saturated fatty acid, was increased by 1.43 fold (fig. 3).
Example 7: effect of enhanced Weissella mesenteroides LCW-28 and ZQ-01 on flavor content
The volatile flavor substances are measured by adopting a solid phase micro-extraction combined gas chromatography-mass spectrometry (SPME-GC-MS) technology, and the specific method refers to the literature. Taking three parallel samples from each sample at the end of fermentation, filtering, mixing uniformly, accurately measuring 5mL of soy sauce sample, placing in a 20mL headspace sample injection bottle, adding 83.36 μ g.L-1Taking 2-octanol as internal standard, mixing uniformly, immediately sealing, and carrying outAnd (4) measuring. And (3) comparing and identifying the mass spectrogram of the sample with a NIST2.0 standard spectrum library after determination. Volatile substances were characterized according to Retention Index (RI). The volatile substances were quantitatively analyzed according to the area of the internal standard 2-octanol.
The reinforced Weissella mesenteroides or the Zygosaccharomyces rouxii can obviously increase the content of main flavor substances (alcohol, acid, ester, phenol, aldehyde and the like) in the soy sauce fermentation process, and particularly, the content of the flavor substances is increased most when the two bacteria are added together. Compared with the Control group, the total content of volatile flavor substances is increased by 2.12 times when the two bacteria are enhanced. Wherein the alcohol is increased by 1.39 times, the acid is increased by 2.39 times, and the aldehyde is increased by 2 times. Wherein the characteristic flavor substance of the soy sauce is 4-ethylguaiacol is improved by 1.58 times, and the HEMF is improved by 2.73 times.
TABLE 3 Main differential volatile substance content (g/kg) in different soy sauce samples
TABLE 4 content of major volatile substances (g/kg) in different soy sauce samples
Comparative example: the influence of the joint reinforcement of different Weissella bacteria and ZQ-01 on the flavor substance content of the sauce mash sample
Soy sauce was prepared by fortifying the other Weissella bacteria selected in example 1, such as Weissellactonusa fusi (panel 1) and Weissella civora Weissellacia Weissellaspora (panel 2), with ZQ-01, respectively, in the same manner as in examples 2 and 7. As shown in Table 5, the other two Weissella strains screened in example 1 were selected for co-fortification with ZQ-01, which was a zygosaccharomyces rouxii strain with a flavor content about 50% less than the co-fortification of Weissella mesenteroides LCW-28 with ZQ-01.
TABLE 5 content of major volatile substances (g/kg) in different soy sauce samples
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. Weissella mesenteroides (Weissella parameteroides) LCW-28 is preserved in China center for type culture Collection (CCTCC M2020778) at 11/27 th of 2020, and the preservation address is China, Wuhan university.
2. A starter culture comprising Weissella mesenteroides LCW-28 according to claim 1.
3. A method for increasing the content of flavor substances in soy sauce, which comprises adding Weissella mesenteroides LCW-28 of claim 1 or the leavening agent of claim 2 and ZQ-01 of ZQ.
4. The method of claim 3, wherein the Weissella mesenteroides LCW-28 of claim 1 or the starter culture of claim 2 is present in the fermentation system at a final concentration of 1.0 x 106～1.0×108CFU/g。
5. The method of claim 3, wherein the ZQ-01 and the Weissella mesenteroides of claim 1 or the starter of claim 2 are added in a ratio of 1:1.
6. The method for preparing the leaven according to claim 2, wherein the Weissella mesenteroides LCW-28 of claim 1 is activated, the bacteria liquid obtained after activation is centrifuged, the precipitate is collected, the buffer solution and the cryoprotectant are added into the precipitate to obtain a re-suspension, and the re-suspension is subjected to vacuum freeze drying to obtain the leaven.
7. The method according to claim 6, wherein the buffer solution is 0.1-0.3M phosphate buffer solution and/or 0.5-1% physiological saline and/or double distilled water, and the cryoprotectant is 10-20% glycerol and/or 8-12% trehalose.
8. Use of the weissella mesenteroides of claim 1 or the starter culture of claim 2, or the process of any one of claims 3 to 5 for fermenting soy sauce.
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