CN114107138A - Combined microbial inoculum for fermented food and application thereof - Google Patents
Combined microbial inoculum for fermented food and application thereof Download PDFInfo
- Publication number
- CN114107138A CN114107138A CN202111638299.XA CN202111638299A CN114107138A CN 114107138 A CN114107138 A CN 114107138A CN 202111638299 A CN202111638299 A CN 202111638299A CN 114107138 A CN114107138 A CN 114107138A
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- CN
- China
- Prior art keywords
- broad bean
- fermented
- microbial inoculum
- candida
- fermentation
- Prior art date
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Abstract
The invention discloses a combined microbial inoculum for fermented food and application thereof, belonging to the technical field of microorganisms. The invention designs a compound fermentation microbial inoculum suitable for Pi county broad bean paste fermented grains, sequentially inoculates pediococcus acidilactici, staphylococcus carnosus and candida fragrans in equal proportion and sequence, so that the amount of flavor compounds generated by the Pi county broad bean paste fermentation can reach 63.1% of that of in-situ fermentation, the types of the flavor compounds are similar, the level of amino acid nitrogen reaches 0.45g/100g, and is far beyond 0.3g/100g specified in GB 2718-.
Description
Technical Field
The invention relates to a combined microbial inoculum of fermented food and application thereof, belonging to the technical field of microorganisms.
Background
For thousands of years, fermented foods have become an important component of the world's dining table. The fermented food is involved in the assembly of intestinal microbiome by providing vitamins, minerals, calories and other nutrients, etc., and converts the substrate into a biologically active or bioavailable end product, some of which have antihypertensive, anticancer, anti-inflammatory, antidiabetic, antithrombotic and antiatherogenic properties. However, most fermented foods are open-fermented, and an important feature of this fermentation is that it is not controllable. This leads to a number of problems, for example environmental microbial invasion during fermentation leading to the production of harmful compounds. The introduction of the artificially constructed combined microbial inoculum can possibly solve a series of problems of product uniformity, safety and the like.
At present, the Pixian broad bean paste fermentation inoculant is mostly used in the starter propagation stage, and flavor compounds are more fully generated from the perspective of providing substrates by adding auxiliary raw materials for degradation in the inoculant. However, pi county broad bean mash is prepared by mixing a koji and saline water and fermenting, and after mixing, many microorganisms contained in the koji cannot adapt to a high-salt environment. Therefore, in the fermentation process of the Pi county broad bean paste fermented grains, most of microorganisms directly having direct effect on flavor are environmental microorganisms, and the environmental microorganisms are influenced by various environmental factors, so that the instability of the environmental microorganisms entering the Pi county broad bean paste fermented grains causes the instability of the finally fermented broad bean paste fermented grains and is susceptible to harmful substances.
The traditional fermented food industry represented by the Pi county broad bean paste faces industrial transformation, and an important characteristic in the transformation process is that a fixed fermentation device and a fermentation microbial inoculum are used for fermentation so as to ensure the stability of the product. Therefore, the development of a fermentation inoculant suitable for the Pixian bean paste is urgently needed.
Disclosure of Invention
In order to solve the problems of harmful microorganism invasion and harmful substance generation caused by natural fermentation of fermented food at present, the invention improves the naturally exposed fermented microbial inoculum of the fermented food, and provides a synthetic microbial inoculum which is sequentially inoculated with pediococcus acidilactici, staphylococcus carnosus and candida changensis in equal proportion and is rationally constructed from bottom to top.
The invention provides a combined microbial inoculum which contains pediococcus acidilactici, staphylococcus carnosus and candida variabilis.
In one embodiment, the combined microbial inoculum comprises pediococcus acidilactici, staphylococcus carnosus and candida variabilis in a ratio of 1:1: 1.
In one embodiment, the number of pediococcus acidilactici, staphylococcus carnosus and candida variabilis in the combined microbial inoculum is more than or equal to 1 × 107CFU/mL or more than or equal to 1X 107CFU/g。
In one embodiment, the combined microbial inoculum is obtained by culturing pediococcus acidilactici, staphylococcus carnosus and/or candida changensis in a synthetic medium; the synthetic medium contains: 6g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 4.5g/L glucose, 1.98g/L fructose, 0.91g/L mannitol, 1.8g/L arabinose, 0.84g/L threonine, 0.80.84g/L isoleucine, 1.38g/L leucine, 0.81g/L phenylalanine, 0.51g/L glycine, 1.06g/L alanine, 1.53g/L lysine, 0.06g/L histidine, 1.73g/L arginine, 0.25g/L methionine, 0.15g/L serine, 0.81g/L proline, 0.98g/L valine, 2.95g/L glutamic acid, 1.76g/L aspartic acid, 0.85g/L tyrosine, 0.51g/L glutamine, 0.02g/L cysteine, L, EDTA 50mg/L tyrosine, 0.02g/L, 8.3mg/L ferric chloride hexahydrate, 0.84mg/L zinc chloride, 0.13mg/L copper chloride dihydrate, 0.1434mg/L cobalt chloride hexahydrate, 0.1mg/L boric acid, 0.016mg/L manganese chloride tetrahydrate, 1mg/L ammonium sulfate hydrochloride, 1mg/L riboflavin, 1mg/L nicotinic acid, 1mg/L calcium pantothenate, 2mg/L pyridoxine, 10mg/L biotin, 1mg/L folic acid, 10mg/L p-aminobenzoic acid, 1M CaCl2 1mg/L、1M MgSO4 0.3mg/L。
The invention also provides application of the combined fungicide in improving the flavor of Pixian broad bean paste.
In one embodiment, the pediococcus acidilactici, the staphylococcus carnosus and the candida variabilis are sequentially inoculated into the Pi county broad bean mash to be fermented and fermented at 25-35 ℃ for at least 25 days.
In one embodiment, the application is to sequentially treat pediococcus acidilactici, staphylococcus carnosus and candida variabilis at a ratio of more than or equal to 1 x 107CFU/gPi county broad bean mashInoculating the inoculated amount of the strain into Pi county broad bean paste to be fermented, and fermenting for 25-35 days at 28-30 ℃.
In one embodiment, the application is to treat Pediococcus acidilactici at ≥ 1 × 107CFU/gPi county broad bean mashInoculating the inoculated amount of the staphylococcus aureus to Pi county broad bean mash to be fermented, and fermenting for 7 days, and then adding the staphylococcus carnosus at a ratio of more than or equal to 1 multiplied by 107CFU/gPi county broad bean mashInoculating the inoculation amount of the candida antalochia to the Pi county broad bean mash to be fermented, continuing to ferment for 7 days, and then adding more than or equal to 1 multiplied by 107CFU/gPi county broad bean mashInoculating the inoculated amount of the strain to Pi county broad bean paste mash to be fermented and continuing to ferment.
In one embodiment, the application is to treat Pediococcus acidilactici at ≥ 1 × 107CFU/gPi county broad bean mashInoculating the strain to fermented soybean grains of Pixian broad bean, fermenting at 30 deg.C for 7 days, and adding Staphylococcus carnosus at a ratio of 1 × 107CFU/gPi county broad bean mashInoculating the inoculation amount of the candida antarctica to Pi county broad bean mash to be fermented, continuing to ferment for 7 days at the temperature of 30 ℃, and then adding more than or equal to 1 multiplied by 10 candida antarctica7CFU/gPi county broad bean mashInoculating the inoculated amount of the strain into Pi county broad bean paste to be fermented, and continuously fermenting at 30 ℃ for 30 days in total.
The invention also provides application of the combined microbial inoculum in improving fermentation seasoning.
In one embodiment, the application includes, but is not limited to, adding the combined microbial inoculum to a soy sauce mash to be fermented.
Has the advantages that: the invention establishes a method for constructing a synthetic microorganism group from bottom to top on the basis of the characteristics of single bacteria. Has the following advantages: (1) realizing the rational construction strategy of the synthetic microbiome from bottom to top; (2) the synthetic microorganism group can be manipulated, and can be directionally combined with double bacteria according to the characteristics of single bacteria to obtain the characteristics of ideal flora; (3) can be popularized, can be used in other fermented food fields, and improves the actual production.
Based on the method, the compound fermentation inoculant suitable for the Pi county broad bean mash is designed, the amount of flavor compounds generated by the fermentation inoculant in a solid state fermentation experiment averagely reaches 63.1% of that of in-situ fermentation, the flavor structure shows higher similarity with natural fermentation, and meanwhile, compared with an in-situ fermentation system, the level of amino acid nitrogen of the inoculant is higher and averagely reaches 0.45g/100g, which is far beyond 0.3g/100g specified by GB 2718-plus 2014. In addition, the combined microbial inoculum also has certain characteristics, and compared with an in-situ system, the combined microbial inoculum generates 9 specific flavor compounds, including Carbon dioxide, 2,3-Butanediol and the like.
Drawings
Fig. 1 shows a selection strategy and a method of target aroma-producing microbial genera and microbial species of fermented soybean paste in Pixian county.
Fig. 2 shows the growth conditions of target microorganisms of Pi county broad bean paste mash under different pH and temperature.
FIG. 3 shows the flavor generation by fermentation of the two-bacterium synthetic flora. (A) Analyzing main coordinates of different flora scales; (B) comparing the flavor quantity generated by a double-bacterium synthetic flora consisting of the candida variabilis and the pediococcus acidilactici with that generated by a flora member; (C) the flavor composition of the candida changensis and pediococcus acidilactici double-bacterium synthetic flora; (D) analyzing main coordinates of different double-bacterium synthetic inoculation sequences; (E) counting the flavor of different inoculation sequences of a double-bacterium synthetic flora consisting of candida changensis and pediococcus acidilactici; (F) the flavor components of the dual-bacterium synthetic flora consisting of the candida changensis and the pediococcus acidilactici in different inoculation sequences.
FIG. 4 is a diagram showing the interaction of two microorganisms.
FIG. 5 shows the flavor profile of the bacterial flora on different scales. (A) Counting the number of flavors generated by different flora scales; (B) the flavor type of the blank control; (C) the flavor variety of the zygosaccharomyces rouxii 2.1522 generated by single bacterium; (D) the zygosaccharomyces rouxii 2.1522 and the flavor species generated by the pediococcus acidilactici; (E) the flavor types generated by the three bacteria synthetic floras of zygosaccharomyces rouxii 2.1522, pediococcus acidilactici and staphylococcus carnosus.
FIG. 6 is a flavor comparison of solid state simulated fermentation and in situ fermentation of a combined microbial inoculum; (A) combining the number of flavor compounds of the microbial inoculum simulated fermentation and in-situ fermentation samples; (B) common and special flavor Wien diagrams of combined microbial inoculum simulated fermentation and in-situ fermentation samples; (C) solid-state simulated fermentation, blank control and in-situ fermentation system pH of the combined microbial inoculum; (D) the combined microbial inoculum solid state simulation fermentation, blank contrast and an in-situ fermentation system amino acid nitrogen content histogram.
Detailed Description
Example 1 microbial screening of combination inocula
The flavor components of the natural fermentation samples were first used as evaluation criteria. Different screening conditions were used to select target strains from a complex community. Then, single-strain fermentations were performed in total synthetic medium to determine the characteristics of the different strains. And performing double-strain fermentation according to the functions of the strains. Microbial communities with three or more microorganisms are established according to the functional performance of single and double strain fermentations and the interaction of microorganisms. Solid state fermentation is carried out to evaluate the actual fermentation effect of the synthetic flora, and finally the combined microbial inoculum inoculated by pediococcus acidilactici, staphylococcus carnosus and candida changensis in sequence in equal proportion is constructed. The method comprises the following specific steps:
(1) target microorganism species selection and design of synthetic media
And taking the relative abundance, the frequency and the feature vector center as screening indexes of the target microorganism genus. In order to select important microbial species in a target genus of microorganisms, we investigated whether or not there are microbial species/strains widely used in industrial production of the target genus of microorganisms, and if there is any, the genus is represented by the species/strain, and if not, the corresponding species is selected based on the alignment result of the OTU sequence most abundant in the target genus. Then, according to GRAS and related reports, pathogenic microorganisms were filtered to obtain core microorganisms potentially important in flavor development in the fermented soybean mash of bean paste of bean of pi county (fig. 1), including candida mutabilis, pediococcus acidilactici, pediococcus pentosaceus, staphylococcus carnosus, weissella fusca, zygosaccharomyces rouxii, pseudomonas paracasei, and halomonas variabilis.
In addition, due to factors such as instability, easy pollution and long fermentation period of an in-situ fermentation system, a fully synthetic culture medium (table 1) which can be carried out in a laboratory and meets the growth and metabolism of all target microorganisms is designed by referring to the nutrient composition of in-situ fermentation, and subsequent experiments are carried out on the basis of the culture medium.
TABLE 1 synthetic Medium formulation
Note: filtering with 0.22 μm filter membrane for sterilization, and adjusting pH to 5.0 with dilute hydrochloric acid and sodium hydroxide solution
(2) Individual character of target microorganism
The fermentation conditions based on the total synthetic medium experiments were determined by considering the growth state of the target microorganism and the in situ environmental conditions (normal temperature, pH 5.0-5.5). Fermenting screened target microorganisms (Candida mutabilis, Pediococcus acidilactici, Pediococcus pentosaceus, Staphylococcus carnosus, Weissella fusca, Zygosaccharomyces rouxii, Pseudomonas paracasei and Halomonas variabilis) on synthetic culture medium respectively, dividing into 20 groups according to culture conditions, fermenting for 48 hours under culture conditions of pH 5.0, 5.5, 6.0, 6.5, 7.0, temperature 30 ℃, 37 ℃, 42 ℃ and 50 ℃, and fermenting according to OD of fermentation liquor600The growth of the microorganisms was evaluated. As shown in FIG. 2, pH (5.0-7.0) and temperature (30-37 ℃) had little effect on microbial growth. But when the temperature reaches 50 ℃, the growth of the microorganism is inhibited. Therefore, conditions (pH 5.0, T30 ℃) under which the microorganisms exhibited similar growth states were selected as fermentation conditions for the following experiments.
The characteristics of the microbial strains constituting the synthetic microbiome are the basis of subsequent studies. Therefore, the composition of volatile compounds produced by the target microorganism was examined as follows.
Extracting flavor substances in the sample by using HS-SPME-Arrow: 1.5g NaCl was weighed, and 5mL of the sample was stirred with ultrapure water to 10% by volume in a headspace bottle and sealed with a hollow metal cap of a PTFE/blue silica gel pad. The headspace solid phase microextraction was performed using a CTC multifunctional automated sample injection system (PAL). The extraction process is as follows: a120-micron DVB/CA R/PDMS three-phase extraction head is adopted, the extraction temperature is 50 ℃, the sample balance time is 5min, the extraction time is 50 min, and the distiller time is 500 min/min. And (4) after extraction is finished, performing desolventizing on the extraction head at a GC injection port for 5 minutes at 250 ℃, and performing GC-MS detection analysis. GC conditions were as follows: the sampling port and the thermometer at 250 deg.C, carrier gas flow rate of 1mL/min, sampling mode of no-flow sampling, and column of DB-FFAP (60m × 0.32mm × 0.25 μm). Baking procedure: after the temperature is kept at 40 ℃ for 2 minutes, the temperature is increased to 150 ℃ at the speed of 4 ℃/minute, the temperature is kept for 2 minutes, and then the temperature is decreased to 230 ℃ at the speed of 6 ℃/minute, and the temperature is kept for 5 minutes. MS conditions: the EI ionization source is used as an ion source, the ionization energy is 70eV, the ion source temperature is 230 ℃, and the scanning range is 35.00-350.00 u.
TABLE 2 content of flavor compound produced by single-strain fermentation of Pixian bean cotyledon with target aroma-producing microorganism
The results show (table 2). As shown by the clustering results of volatile compounds, different species of target microorganisms produce interception of different volatile compounds. Among them, yeast mainly produces alcohol compounds, while lactic acid bacteria produce more acetic acid and a small amount of related acetate esters. Other microorganisms can produce some aldehydes, such as acetaldehyde, 2-methylbutyraldehyde, etc., which are not detectable in yeast and lactic acid bacteria. Thus, depending on the composition of the volatile substance, microorganisms can be divided into three categories: yeast, lactic acid bacteria and others, and there is a clear complementary relationship between volatile substances of different species of microorganisms.
(3) Effect of flora Scale and order of inoculation of the two-bacterium synthetic flora on the production of flavor Compounds
The complementary relation between the fragrance production of different kinds of microorganisms provides a theoretical basis for the rational design of a high-dimensional microorganism group. Following this complementary relationship, microorganisms belonging to different species were randomly selected in pairs and inoculated for fermentation (in OD) using fully synthetic medium in different inoculation sequences600The bacterial solution (0.7) was inoculated into the synthetic medium at an inoculum size of 2%, the interval between two microbial inoculations was 48 hours, other conditions were the same as for single-strain fermentations), and the total time for each fermentation was 6 days.
As shown in FIG. 3A, the single-bacterium fermentation of Pediococcus acidilactici and Zygosaccharomyces rouxii and the combination of two bacteria with different inoculation sequences are taken as examples and specifically grouped as follows:
single-bacterium fermentation: culturing Pediococcus acidilactici or Zygosaccharomyces rouxii to OD600Inoculating the bacterial liquid of which the inoculation amount is 0.7 to a synthetic culture medium according to the inoculation amount of 2 percent, and respectively naming the bacterial liquid as a single bacterium-pediococcus acidilactici group and a single bacterium-zygosaccharomyces rouxii group;
double-bacterium fermentation: culturing Pediococcus acidilactici or Zygosaccharomyces rouxii to OD600Inoculating the bacterial liquid which is 0.7 to a synthetic culture medium according to the inoculation amount of 2 percent and a certain inoculation sequence, wherein:
dual bacteria-post yeast group: firstly inoculating pediococcus acidilactici, culturing for 48h, and then inoculating zygosaccharomyces rouxii;
two-first inoculation yeast group: inoculating zygosaccharomyces rouxii, culturing for 48h, and inoculating Pediococcus acidilactici;
dual bacteria-simultaneous inoculation: pediococcus acidilactici and Zygosaccharomyces rouxii were inoculated at a ratio of 1: 1.
Comparison of the flavour development of the different scale populations showed significant differences. The blank control group had the lowest aroma-producing capacity, followed by single-strain fermentation, and the highest amount of flavor compounds produced by double-strain fermentation (fig. 3B). Furthermore, as shown in fig. 3D, the aroma production of the combination of two bacteria, which are members of the same flora but different inoculation sequences, showed significant differences (e.g. in the case of the combination of zygosaccharomyces rouxii and pediococcus acidilactici, two single cultures produced 13 and 14 volatile compounds, respectively (fig. 3B, see table 2 for details), whereas in the combination of two bacteria, the number of flavors produced by inoculating yeast first or inoculating staphylococcus first and then yeast first was as high as 24, while the number of aromas produced by inoculation was lower but higher than that of a single bacteria (fig. 3E)), the combination of two bacteria produced some unique flavor compounds that could not be produced by the experimental group of single bacteria, such as 2, 3-butanedione, dimethyldisulfide, etc. These compounds accounted for 39% of the total amount of biflavone flavour compounds (fig. 3C), while flavour compounds specific to different inoculation sequences accounted for 46% of the biflavone flavour (fig. 3F).
(4) Microbial interaction
To study the interaction between different microorganisms, experiments on microbial interaction were performed on solid synthetic media as shown in FIG. 4. The experimental result shows that in all target microorganisms, the yeast can inhibit the growth of other microorganisms, and occupies absolute growth advantage, and the staphylococcus is inferior, so that the lactic acid bacteria has the weakest competitive power when being co-cultured with other microorganisms. Therefore, the ideal inoculation sequence would be to inoculate the lactic acid bacteria first, then the staphylococci and finally the yeast. Thereby promoting the growth and metabolism of different microorganisms in the same system to the maximum extent to generate more flavor compounds.
Example 2 Combined solid State fermentation Using three bacteria microorganisms
In order to construct a synthetic microorganism group which can generate more flavor compounds and is closer to the flavor composition of an in-situ system, three bacteria combinations inoculated with lactococcus lactis-staphylococcus carnosus-candida changensis in a specific sequence are designed on the basis of single-bacteria fermentation, double-bacteria fermentation and double-bacteria interaction, and the pediococcus lactis, the staphylococcus carnosus and the candida changensis are respectively cultured to OD600Culturing to OD of 2%600Inoculating 0.7 Pediococcus acidilactici bacterial solution to synthetic culture medium, culturing for 48 hr, inoculating 2% of the bacterial solution, and culturing to OD6000.7 of the staphylococcus carnosus liquid, culturing for 48h, and inoculating with 2% of the staphylococcus carnosus liquidSeed culture to OD600And (3) fermenting the candida changensis strain solution for 6 days, wherein the volatile substance detection result shows that the flavor quantity generated by the microorganism group is increased along with the enlargement of the scale of the synthetic microorganism group, and the difference of the flavor quantity between the parallel is gradually reduced. In addition, as the size of the synthetic microbiome increased, the microbiome produced more uniform flavor profiles and a greater number of species (fig. 5).
The combined microbial inoculum of three microorganisms of lactococcus lactis-staphylococcus carnosus-candida changensis is used for solid state fermentation of the Pi county broad bean paste. In order to evaluate the effect of the 3 bacteria combination in a solid state fermentation system, the solid state fermentation is carried out by using a microbial inoculum containing the 3 bacteria combination in a laboratory horizontal simulation in-situ system process, and lactococcus lactis-staphylococcus carnosus-pseudomyceliophthora changensis is inoculated in sequence at 30 ℃, a microorganism is inoculated every 7 days, and the inoculation amount is 1 multiplied by 107cfu/gPi county broad bean mashNamely, the fermented Pi county broad bean mash is used as a raw material to be inoculated with 1 multiplied by 107cfu/gPi county broad bean mashLactococcus lactis, fermented at 30 ℃ for 7 days and inoculated with 1X 107cfu/gPi county broad bean mashFermenting Staphylococcus carnosus at 30 deg.C for 7 days, inoculating with 1 × 107cfu/gPi county broad bean mashThe Candida was transformed for 30 days. The raw materials for in-situ fermentation are used as blank control, and the in-situ fermentation is to mix the fermented grains of the Pixian broad beans with yeast and ferment the mixture for 30 days at 30 ℃ by adopting the original process.
TABLE 3 content of flavor Compounds in samples fermented with combination of microbial inoculum and in situ fermentation
Compared with the sample of Pi county broad bean paste fermented grain naturally fermented in situ, the combined microbial inoculum simulated solid-state fermentation generates 39-44 volatile compounds (table 3), the total amount of the volatile compounds accounts for 63.1% of the amount of the volatile compounds in situ (figure 6A), and the flavor types and the in situ fermentation result show higher similarity. In order to research the specific fermentation effect of the 3-bacterium combination, the specific difference between the volatile compounds fermented by the 3-bacterium group and the volatile compounds fermented in situ is compared. 35 compounds were detected in both samples, including 1-octen-3-ol, 3-methylthiopropanol, guaiacol, etc., which were identified as important flavour compounds in Pixian bean paste. The synthetic microbiome fermentation specifically produced 9 volatile compounds including carbon disulfide, 2,3-butanediol, etc. (FIG. 6B).
On the other hand, amino acid nitrogen is an important industrial index, so that the difference of the amino acid nitrogen content of 3 bacterial flora and an in-situ fermentation sample is compared. As shown in fig. 6D, the content of amino acid nitrogen in fermented broad bean paste of pi county broad bean after the combined microbial inoculum fermentation averagely reaches 0.45g/100g, which is significantly higher than that of the blank control group and the in-situ fermentation group. Although this may be related to the difference in the raw materials used and the scale of fermentation, it was also found by comparison with the blank control group that the combination fungicide fermentation exhibited excellent amino acid nitrogen levels. In addition, the difference between the pH of the combination inoculum, the blank control and the pH of the pi county broad bean mash fermented in situ may explain to some extent the difference in the level of amino acid nitrogen (fig. 6C). Due to the introduction of a large amount of environmental microorganisms, a large amount of acid is generated in an in-situ fermentation system, and the metabolic activity of microorganisms related to protein degradation and amino acid metabolism is inhibited while inhibiting part of harmful microorganisms, so that the level of amino acid nitrogen is relatively reduced. The combined microbial inoculum only contains 3 microorganisms, so that the microorganisms produce less acid, the related metabolic activity is not inhibited by a weak acid environment, and the combined microbial inoculum shows a higher level of amino acid nitrogen.
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 (10)
1. The combined microbial inoculum is characterized by comprising pediococcus acidilactici, staphylococcus carnosus and candida variabilis; the number ratio of pediococcus acidilactici, staphylococcus carnosus and candida variabilis is 1:1: 1.
2. The combination microbial inoculum of claim 1, wherein the number of pediococcus acidilactici, staphylococcus carnosus and candida variabilis in the combination microbial inoculum is more than or equal to 1 x 107CFU/mL or more than or equal to 1X 107CFU/g。
3. The combination microbial inoculum according to claim 2, which is prepared by mixing bacterial liquids obtained by respectively culturing pediococcus acidilactici, staphylococcus carnosus and candida variabilis.
4. The combination microbial inoculum according to claim 2, which is obtained by culturing pediococcus acidilactici, staphylococcus carnosus and/or candida changensis in a synthetic medium; the synthetic medium contains: 6g/L disodium hydrogen phosphate, 3g/L potassium dihydrogen phosphate, 4.5g/L glucose, 1.98g/L fructose, 0.91g/L mannitol, 1.8g/L arabinose, 0.84g/L threonine, 0.80.84g/L isoleucine, 1.38g/L leucine, 0.81g/L phenylalanine, 0.51g/L glycine, 1.06g/L alanine, 1.53g/L lysine, 0.06g/L histidine, 1.73g/L arginine, 0.25g/L methionine, 0.15g/L serine, 0.81g/L proline, 0.98g/L valine, 2.95g/L glutamic acid, 1.76g/L aspartic acid, 0.85g/L tyrosine, 0.51g/L glutamine, 0.02g/L, EDTA 50mg/L cysteine, 8.3mg/L ferric chloride hexahydrate, 0.84mg/L zinc chloride, 0.13mg/L copper chloride dihydrate, 0.1434mg/L cobalt chloride hexahydrate, 0.1mg/L boric acid, 0.016mg/L manganese chloride tetrahydrate, 1mg/L ammonium sulfate hydrochloride, 1mg/L riboflavin, 1mg/L nicotinic acid, 1mg/L calcium pantothenate, 2mg/L pyridoxine, 10mg/L biotin, 1mg/L folic acid, 10mg/L p-aminobenzoic acid, 1M CaCl2 1mg/L、1M MgSO4 0.3mg/L。
5. Use of the combined fungicide according to any one of claims 1 to 4 for improving the flavor of Pixian bean paste.
6. A method for improving the flavor of a Pi county broad bean paste is characterized in that pediococcus acidilactici, staphylococcus carnosus and candida fragrans are sequentially inoculated into the Pi county broad bean paste to be fermented and fermented at 25-35 ℃ for at least 25 days.
7. The method according to claim 6, wherein Pediococcus acidilactici, Staphylococcus carnosus and Candida variabilis are sequentially treated at a ratio of 1X 10 or more7CFU/gPi county broad bean mashInoculating the inoculated amount of the strain into Pi county broad bean paste to be fermented, and fermenting for 25-35 days at 28-30 ℃.
8. The method of claim 7, wherein Pediococcus acidilactici is administered at a rate of 1X 10 or more7CFU/gPi county broad bean mashInoculating the strain to Pi county broad bean mash to be fermented, fermenting for 7 days, and then adding staphylococcus carnosus at a ratio of more than or equal to 1 × 107CFU/gPi county broad bean mashInoculating the inoculation amount of the candida antalochia to the Pi county broad bean mash to be fermented, continuing to ferment for 7 days, and then adding more than or equal to 1 multiplied by 107CFU/gPi county broad bean mashInoculating the inoculated amount of the strain to Pi county broad bean paste mash to be fermented and continuing to ferment.
9. The application of the combined microbial inoculum of any one of claims 1 to 4 in improving the content of flavor substances in fermented seasonings.
10. The use according to claim 9, wherein the use includes, but is not limited to, adding the combined microbial inoculum to a sauce mash of a seasoning to be fermented.
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