CN114107138B - Combined microbial agent for fermented food and application thereof - Google Patents

Combined microbial agent for fermented food and application thereof Download PDF

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CN114107138B
CN114107138B CN202111638299.XA CN202111638299A CN114107138B CN 114107138 B CN114107138 B CN 114107138B CN 202111638299 A CN202111638299 A CN 202111638299A CN 114107138 B CN114107138 B CN 114107138B
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徐岩
张丽杰
郑鹏飞
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Sichuan Pixian Douban Co ltd
Jiangnan University
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Jiangnan University
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Abstract

The invention discloses a combined microbial agent for fermented foods and application thereof, and belongs to the technical field of microorganisms. The invention designs a composite fermentation inoculant suitable for Pi county broad bean paste fermented grains, and by sequentially inoculating Pediococcus acidilactici, staphylococcus botulinum and candida mutans in equal proportion, the amount of flavor compounds generated by Pi county broad bean paste fermentation can reach 63.1% of in-situ fermentation, the types of the flavor compounds are similar, and the amino acid nitrogen level reaches 0.45g/100g and is far more than 0.3g/100g specified in GB 2718-2014.

Description

Combined microbial agent for fermented food and application thereof
Technical Field
The invention relates to a combined microbial agent for fermented foods and application thereof, and belongs to the technical field of microorganisms.
Background
Fermented foods have become an important component of global dining tables for thousands of years. Fermented foods are produced by providing vitamins, minerals, calories and other nutrients to participate in the assembly of the intestinal microbiome and converting the substrate into a biologically active or bioavailable end product, some of which have antihypertensive, anticancer, anti-inflammatory, antidiabetic, antithrombotic and antiatherosclerotic functions. However, most fermented foods are open-ended, and this mode of fermentation is not controllable as an important feature. This results in a number of problems, such as the invasion of environmental microorganisms 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 uniformity, safety and the like of products.
The existing Pi county bean cotyledon fermentation microbial inoculum is mainly used in the starter propagation stage, and the microbial inoculum is added to assist in raw material degradation, so that the flavor compound is more fully generated from the standpoint of providing a substrate. However, the Pi county broad bean paste is prepared by mixing and fermenting yeast material and brine, and most of microorganisms contained in the yeast material cannot adapt to the high-salt environment after mixing. Therefore, in the process of fermenting the Pi county broad bean paste fermented grains, most of microorganisms directly acting on the flavor are from 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 leads to the instability of the final fermented broad bean paste fermented grains and the easy infection of harmful substances.
The traditional fermented food industry represented by Pi county bean cotyledon is facing industrial transformation, and an important characteristic in the transformation process is to use a fixed fermentation device and a fermentation microbial inoculum for fermentation so as to ensure the stability of the product. Therefore, development of a fermentation starter suitable for Pi county bean cotyledon is needed.
Disclosure of Invention
In order to solve the problems of harmful microorganism invasion and harmful substance generation caused by natural fermentation of the fermented food at present, the invention provides a synthetic microbial agent which is reasonably constructed from bottom to top and is inoculated with Pediococcus acidilactici, staphylococcus botulinum and candida mutans in equal proportion by improving the natural exposure fermentation microbial agent of the fermented food.
The invention provides a combined microbial inoculum which contains Pediococcus acidilactici, staphylococcus botulinum and candida mutans.
In one embodiment, the combined microbial agent comprises Pediococcus acidilactici, staphylococcus botulinum and Candida variabilis in a number ratio of 1:1:1.
In one embodiment, the amount of Pediococcus acidilactici, staphylococcus botulinum and Candida variabilis in the combined microbial inoculum is not less than 1×10 7 CFU/mL or ≡1X10. 7 CFU/g。
In one embodiment, the combination of bacteria is a combination of Pediococcus acidilactici, staphylococcus botulinum and/or Candida mutans inCulturing in a synthetic medium; the synthetic medium contains: disodium hydrogen phosphate 6g/L, potassium dihydrogen phosphate 3g/L, glucose 4.5g/L, fructose 1.98g/L, mannitol 0.91g/L, arabinose 1.8g/L, threonine 0.84g/L, isoleucine 0.80.84g/L, leucine 1.38g/L, phenylalanine 0.81g/L, glycine 0.51g/L, alanine 1.06g/L, lysine 1.53g/L, histidine 0.06g/L, arginine 1.73g/L, methionine 0.25g/L, serine 0.15g/L, proline 0.81g/L, valine 0.98g/L, glutamic acid 2.95g/L aspartic acid 1.76g/L, tyrosine 0.85g/L, glutamine 0.51g/L, cysteine 0.02g/L, EDTA mg/L, ferric trichloride hexahydrate 8.3mg/L, zinc chloride 0.84mg/L, cupric chloride dihydrate 0.13mg/L, cobalt chloride hexahydrate 0.1434mg/L, boric acid 0.1mg/L, manganese chloride tetrahydrate 0.016mg/L, ammonium sulfate hydrochloride 1mg/L, riboflavin 1mg/L, niacin 1mg/L, calcium pantothenate 1mg/L, pyridoxine 2mg/L, biotin 10mg/L, folic acid 1mg/L, p-aminobenzoic acid 10mg/L, 1M CaCl 2 1mg/L、1M MgSO 4 0.3mg/L。
The invention also provides application of the combined microbial inoculum in improving the flavor of Pi county bean cotyledon.
In one embodiment, the application is to sequentially inoculate Pediococcus acidilactici, staphylococcus botulinum and Candida variabilis into the Pi county broad bean paste to be fermented, and ferment at 25-35℃for at least 25 days.
In one embodiment, the use is to sequentially increase Pediococcus acidilactici, staphylococcus botulinum and Candida variabilis by 1×10 or more 7 CFU/g Pi county broad bean paste fermented grains Inoculating the seed quantity into Pi county broad bean paste fermented grains to be fermented, and fermenting for 25-35 days at the temperature of 28-30 ℃.
In one embodiment, the use is to treat Pediococcus acidilactici at a rate of 1X 10 or more 7 CFU/g Pi county broad bean paste fermented grains Inoculating the inoculated amount of the strain into Pi county broad bean paste to be fermented, fermenting for 7 days, and then adding staphylococcus meat to the strain of the broad bean paste at a ratio of more than or equal to 1 multiplied by 10 7 CFU/g Pi county broad bean paste fermented grains Inoculating the seed amount of the strain into Pi county broad bean paste to be fermented, continuously fermenting for 7 days, and then adding candida utilis to the seed amount of the strain to be fermented to be more than or equal to 1 multiplied by 10 7 CFU/g Pi county broad bean paste fermented grains Is inoculated toAnd (5) continuously fermenting the Pi county broad bean paste fermented grains to be fermented.
In one embodiment, the use is to treat Pediococcus acidilactici at a rate of 1X 10 or more 7 CFU/g Pi county broad bean paste fermented grains Inoculating the seed amount to Pi county broad bean paste to be fermented, fermenting at 30deg.C for 7 days, and culturing staphylococcus meat at a ratio of 1×10 or more 7 CFU/g Pi county broad bean paste fermented grains Inoculating the seed amount to Pi county broad bean paste to be fermented, fermenting at 30deg.C for 7 days, and fermenting with candida utilis at a ratio of 1×10 or more 7 CFU/g Pi county broad bean paste fermented grains Inoculating the inoculated amount into Pi county broad bean paste fermented grains to be fermented, and continuously fermenting at 30 ℃ for 30 days.
The invention also provides application of the combined microbial inoculum in improving fermented seasonings.
In one embodiment, the application includes, but is not limited to, adding the combined inoculant to a sauce grain to be fermented.
The beneficial effects are that: the invention establishes a method for constructing a synthetic microbiome from bottom to top based on the characteristics of single bacteria. Has the following advantages: (1) Realizing rational construction strategy of constructing the synthetic microbiome from bottom to top; (2) The synthetic microbiome can be operated, and can be directionally combined with double bacteria according to the single bacteria characteristic to obtain the ideal flora characteristic; (3) The method can be popularized and can be used in other fermented food fields to improve actual production.
Based on the method, the composite fermentation inoculant suitable for the Pi county broad bean fermented grains is designed, the number of the flavor compounds generated in the solid state fermentation experiment by applying the fermentation inoculant provided by the invention is 63.1% of that of in-situ fermentation, the structure of flavor type and natural fermentation show higher similarity, and meanwhile, compared with an in-situ fermentation system, the amino acid nitrogen level of Cheng Jun agents is higher, and the average of the amino acid nitrogen level is 0.45g/100g and far exceeds 0.3g/100g specified in GB 2718-2014. 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 disulfide,2,3-Butanediol and the like.
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FIG. 1 shows the strategy and method for selecting the target aroma-producing microorganism genus and species of Pi county broad bean paste fermented grains.
FIG. 2 shows the growth conditions of the target microorganism of Pi county broad bean paste fermented grains at different pH and temperature.
FIG. 3 shows the fermentation flavor development of the double bacterial synthetic flora. (a) principal coordinate analysis of different flora sizes; (B) Comparing the quantity of flavors generated by the double-fungus synthetic flora and flora members consisting of candida utilis and pediococcus acidilactici; (C) Flavor composition of candida variabilis and pediococcus acidilactici double-bacteria synthetic flora; (D) Main coordinate analysis of different double-bacteria synthesis inoculation sequences; (E) Flavor counts of different inoculation sequences of the double-bacteria synthetic flora consisting of candida mutans and pediococcus acidilactici; (F) The flavor composition of the double-bacteria synthetic flora consisting of candida mutans and pediococcus acidilactici is different in inoculation sequence.
FIG. 4 is a graph of interactions of microorganisms two by two.
FIG. 5 shows the structure of flavor type produced by different scale flora. (a) statistics of the number of flavors produced at different flora scales; (B) a control flavor profile; (C) The flavor type produced by the zygosaccharomyces rouxii 2.1522 single bacterium is constituted; (D) The flavor type produced by the zygosaccharomyces rouxii 2.1522 and the pediococcus acidilactici; (E) The flavor type produced by the three synthetic flora of zygosaccharomyces rouxii 2.1522, pediococcus acidilactici and Staphylococcus botulinum.
FIG. 6 is a comparison of flavors of a combined inoculant solid state simulated fermentation versus an in situ fermentation; (A) The amount of flavor compounds of the combined inoculant simulated fermentation and in situ fermentation samples; (B) Combining a common and special flavor wien diagram of a microbial inoculum simulated fermentation and in-situ fermentation sample; (C) Solid-state simulated fermentation, blank control and in-situ fermentation system pH of the combined microbial inoculum; (D) Solid-state simulated fermentation, blank comparison and in-situ fermentation of the combined microbial inoculum system amino acid nitrogen content histogram.
Detailed Description
EXAMPLE 1 microbial screening of combination microbial agents
First, the flavor component of the natural fermentation sample was used as an evaluation standard. Different screening conditions were used to select the target strain from a complex population. Then, single-strain fermentation was performed in total synthetic medium to determine the characteristics of different strains. And performing double-strain fermentation according to the functions of the strain. Based on the functional performance of the single-strain and double-strain fermentations and the microbial interaction, a microbial community is established with three or more microorganisms. And (3) carrying out solid state fermentation to evaluate the actual fermentation effect of the synthetic flora, and finally constructing a combined microbial inoculum which is inoculated with Pediococcus acidilactici, staphylococcus botulinum and candida utilis in equal proportion. The method comprises the following specific steps:
(1) Design of target microorganism species selection and synthetic Medium
The relative abundance, frequency and eigenvector center are used as screening indexes of the target microorganism genus. In order to select an important microorganism species in the target genus of microorganism, we investigated whether the target genus of microorganism has a microorganism species/strain widely used in industrial production, and if so, the genus is represented by the species/strain, and if not, the corresponding species is selected according to the alignment of the highest abundance OTU sequences in the target genus. Then, according to GRAS and related reports, pathogenic microorganisms were filtered to obtain core microorganisms potentially playing an important role in flavor formation in Pi county broad bean fermented grains (FIG. 1), including Candida mutans, pelarococcus acidilactici, pelarococcus pentosaceus, staphylococcus botulinum, weissella fusogenic, zygosaccharomyces rouxii, pseudomonas paralactis, and Salmonella mutans.
In addition, due to the factors of unstable in-situ fermentation system, easy pollution, long fermentation period and the like, a total synthesis culture medium (table 1) which can be used in a laboratory and meets the growth and metabolism of all target microorganisms is designed by referring to the nutrition composition of in-situ fermentation, and the subsequent experiment is carried out based on the culture medium.
Table 1 synthetic medium formulation
Note that: filtering with 0.22 μm filter membrane, sterilizing, and adjusting pH to 5.0 with dilute hydrochloric acid and sodium hydroxide solution
(2) Single bacterial property of target microorganism
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 to 5.5). Fermenting the target microorganism (Candida variabilis, pediococcus acidilactici, pediococcus pentosaceus, staphylococcus botulinum, weissella fusiformis, zygosaccharomyces rouxii, pseudomonas paralactis, and Salmonella variabilis) obtained by screening on synthetic culture medium, respectively, dividing into 20 groups according to culture conditions, fermenting under culture conditions of pH 5.0,5.5,6.0,6.5, 7.0, temperature 30deg.C, 37deg.C, 42deg.C, and 50deg.C for 48 hr according to OD of fermentation broth 600 The 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 reached 50 c, microbial growth was inhibited. Thus, conditions (ph=5.0, t=30 ℃) under which microorganisms exhibited similar growth states were selected as fermentation conditions for the following experiments.
The nature of the microbial strains that constitute the synthetic microbiome is the basis of subsequent studies. Thus, the composition of the volatile compounds produced by the target microorganism was examined as follows.
HS-SPME-Arrow extracts flavor substances from the samples: 1.5g of NaCl was weighed, 5mL of the mixture was accurately weighed, stirred with ultrapure water until the sample liquor was 10% by volume in a headspace bottle, and sealed with a hollow metal cap of a PTFE/blue silica gel pad. A CTC company multifunctional autosampler (PAL) was used for headspace solid-phase microextraction. The extraction process is as follows: the extraction temperature is 50 ℃ and the sample balancing time is 5min, the extraction time is 50 min, and the distiller is 500 min/min by adopting a 120 mu m DVB/CA R/PDMS three-phase extraction head. After extraction, the extraction head is subjected to GC injection port leaching at 250 ℃ for 5 minutes, and GC-MS detection analysis is carried out. GC conditions: the sample inlet and the detection temperature are 250 ℃ popular, the carrier gas flow rate is 1mL/min, the sample injection mode is non-split sample injection, and the column adopts DB-FFAP (60 m multiplied by 0.32mm multiplied by 0.25 mu m). Baking procedure: after maintaining the temperature at 40 ℃ for 2 minutes, the temperature is raised to 150 ℃ at a speed of 4 ℃/minute, the temperature is maintained for 2 minutes, and then the temperature is reduced to 230 ℃ at a speed of 6 ℃/minute, and the temperature is maintained for 5 minutes. MS conditions: and using an EI ionization source as an ion source, wherein the ionization energy is 70eV, the temperature of the ion source is 230 ℃, and the scanning range is 35.00-350.00 u.
TABLE 2 content of flavor Compound produced by Mono-bacterial fermentation of Pi county bean cotyledon target aroma-producing microorganism
The results show (Table 2). As shown by the clustering results of the volatile compounds, different kinds of target microorganisms produce different volatile compounds. Of these, yeasts produce mainly alcohol compounds, whereas lactic acid bacteria produce more acetic acid and a small amount of the relevant acetate. Other microorganisms can produce aldehydes such as acetaldehyde, 2-methylbutyraldehyde, etc., which are undetectable in yeast and lactic acid bacteria. Thus, microorganisms can be classified into three types according to the composition of volatile substances: yeast, lactobacillus and other types, and there is a clear complementary relationship between the volatile substances of different types of microorganisms.
(3) Effects of flora size and order of inoculation of the bifidogenic synthetic flora on the flavour compounds produced
The complementary relation between the fragrance production of different types of microorganisms provides a theoretical basis for rational design of high-dimension microbiome. Following this complementation, microorganisms belonging to different species are randomly selected in pairs and inoculated fermentation (in OD) using total synthetic medium in different inoculation sequences 600 Bacterial solutions of =0.7 were inoculated to the synthetic medium at an inoculum size of 2%, the interval between two microorganism inoculations was 48 hours, the other conditions were the same as for single-bacteria fermentation), and the total fermentation time per group was 6 days.
As shown in fig. 3A, taking a single-bacterial fermentation of pediococcus acidilactici and zygosaccharomyces rouxii as an example, a double-bacterial combination with different inoculation sequences is composed, and the specific grouping is as follows:
single-fungus fermentation: pediococcus acidilactici or RussellCulturing zygosaccharomyces to OD 600 Bacterial solutions=0.7 were inoculated to the synthetic medium at an inoculum size of 2%, respectively designated as a single bacterium-pediococcus acidilactici group, a single bacterium-zygosaccharomyces rouxii group;
double-bacteria fermentation: pediococcus acidilactici or zygosaccharomyces rouxii is cultured to OD 600 Bacterial solutions=0.7 were inoculated to the synthetic medium in an inoculum size of 2% and in a certain inoculation order, wherein:
double fungus-post yeast group: inoculating Pediococcus acidilactici, culturing for 48 hours, and inoculating zygosaccharomyces rouxii;
double fungus-first yeast group: inoculating zygosaccharomyces rouxii, culturing for 48 hours, and then inoculating pediococcus acidilactici;
double fungus-simultaneous inoculation: pediococcus acidilactici and zygosaccharomyces rouxii were inoculated at a ratio of 1:1.
Comparing the produced flavors of different scale populations showed significant differences. The blank group had the lowest aroma-producing capacity, followed by single-strain fermentation, and the largest amount of flavor compounds was produced by double-strain fermentation (fig. 3B). Furthermore, as shown in fig. 3D, the same flora members but different inoculation sequences exhibited significant differences in the aroma production of the two combinations (e.g. in the case of the combination of combined ruhnia yeast and pediococcus acidilactici, two single cultures produced 13 and 14 volatile compounds, respectively (fig. 3B, see table 2 for details), whereas in the two combinations of bacteria, the number of flavors produced by the first inoculation with yeast or the first inoculation with staphylococcus and then inoculation with yeast was up to 24, while the inoculation produced a smaller number of flavors than the single bacteria (fig. 3E)), the two combinations produced some flavor compounds that the specific single bacteria experimental group could not produce, such as 2, 3-butanedione, dimethyl disulfide, etc. These compounds account for 39% of the total amount of dual fungus flavor compounds (fig. 3C), while flavor compounds specific to different inoculation sequences account for 46% of dual fungus flavor (fig. 3F).
(4) Microbial interaction
To study the interactions between different microorganisms, experiments of microbial interactions were performed on solid synthetic media as shown in fig. 4. Experimental results show that in all target microorganisms, the microzyme can inhibit the growth of other microorganisms, takes absolute growth advantage, and the staphylococcus is inferior, so that the lactobacillus has the weakest competition ability when being co-cultured with other microorganisms. Thus, the ideal inoculation sequence would be to inoculate lactic acid bacteria first, then staphylococci, and finally yeasts. Thereby maximizing the growth and metabolism of different microorganisms in the same system to produce more flavor compounds.
Example 2 application of three-bacteria microorganism combination solid State fermentation
In order to construct a synthetic microbiome which can generate more flavor compounds and is closer to the flavor composition of an in-situ system, a three-bacteria combination of lactococcus lactis, staphylococcus meat and candida mutans inoculated in a specific sequence is designed on the basis of single-bacteria fermentation, double-bacteria fermentation and double-bacteria interaction, and the lactococcus lactis, the staphylococcus meat and the candida mutans are respectively cultured to OD 600 =0.7, first culture to OD at a ratio of 2% 600 Pediococcus acidilactici bacterial liquid with the concentration of 0.7 is inoculated into a synthetic culture medium, and is inoculated and cultured to OD according to the proportion of 2% after being cultured for 48 hours 600 Staphylococcus botulinum fluid =0.7, after 48h additional incubation, inoculated at 2% to OD 600 The volatile matter detection results of the candida utilis solution, which is=0.7, after 6 days of co-fermentation, show that the number of flavors produced by the microbiome is increased and the difference in the number of flavors between the parallel lines is gradually reduced as the scale of the synthetic microbiome is increased. In addition, as the scale of the synthetic microbiome increases, the flavor structure produced by the microbiome is more uniform and the number of species is greater (fig. 5).
The combined microbial inoculum of three microorganisms of lactococcus lactis, staphylococcus botulinum and candida mutans is used for solid state fermentation of soybean fermented grains of Pi county broad bean. To evaluate the effect of the above 3-bacterium combination in a solid state fermentation system, we used the 3-bacterium-containing composition to simulate an in situ system process at laboratory level for solid state fermentation, inoculating lactococcus lactis-staphylococcus meat-candida mutans at 30 ℃ in the order of 1×10 each 7 days, inoculating a microorganism 7 cfu/g Pi county broad bean paste fermented grains Namely, taking Pi county broad bean paste fermented grains for fermentation as raw materials, inoculating 1 multiplied by 10 7 cfu/g Pi county broad bean paste fermented grains Lactococcus lactis was inoculated 1X 10 by fermentation at 30℃for 7 days 7 cfu/g Pi county broad bean paste fermented grains The staphylococcus meat is fermented for 7 days at 30 ℃ and inoculated with 1 multiplied by 10 7 cfu/g Pi county broad bean paste fermented grains Candida mutans is fermented for 30 days. The raw materials for in-situ fermentation are used as blank control, and in the in-situ fermentation, the raw materials are mixed with the fermented soybean grains of Pi county beans and fermented for 30 days at the temperature of 30 ℃ by adopting the original process.
TABLE 3 flavor compound content in samples combining microbial agent fermentation and in situ fermentation
Compared with the in-situ natural fermented Pi county broad bean paste samples, the combined microbial inoculum simulates solid state fermentation to generate 39-44 volatile compounds (Table 3), and the combined microbial inoculum accounts for 63.1 percent of the amount of the in-situ volatile compounds (figure 6A) on average, and the flavor type and the in-situ fermentation result show higher similarity. To investigate the specific fermentation effect of the 3-bacteria combination, specific differences in 3-bacteria group fermentation and in situ fermentation of volatile compounds were compared. 35 compounds were detected in both samples, including 1-octen-3-ol, 3-methylthiopropanol, guaiacol, and the like, which have been identified as important flavor compounds for Pixian bean cotyledon. 9 volatile compounds including carbon disulfide,2,3-butanediol, etc. were specifically produced in the fermentation of the synthetic microbiome (FIG. 6B).
On the other hand, amino acid nitrogen is an important industrial index, so we compared the difference in amino acid nitrogen content between 3 bacterial flora and in situ fermentation samples. As shown in FIG. 6D, the amino acid nitrogen content of the Pixian broad bean fermented grains after the combined microbial inoculum is fermented is 0.45g/100g on average, which is obviously higher than that of a blank control group and an in-situ fermentation group. Although this may be related to the differences in the raw materials used and the scale of fermentation, it was also found by comparison with the blank that the combined microbial agent fermentation exhibited excellent amino acid nitrogen levels. In addition, the difference between the pH of the combined inoculant, the blank and the in situ fermented Pi county broad bean paste might explain to some extent the cause of the difference in amino acid nitrogen levels (fig. 6C). Because of the introduction of a large amount of environmental microorganisms, a large amount of acid is generated in the in-situ fermentation system, and the metabolic activity of microorganisms related to protein degradation and amino acid metabolism is inhibited while partial harmful microorganisms are inhibited, so that the level of amino acid nitrogen is relatively reduced. The combined microbial agent has less acid production by the microorganisms due to the fact that the combined microbial agent has only 3 microorganisms, related metabolic activities are not inhibited by a weak acidic environment, and the combined microbial agent shows higher amino acid nitrogen level.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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 (4)

1. A method for improving Pi county bean cotyledon flavor is characterized by comprising mixing Pediococcus acidilactici with a content of 1×10 or more 7 CFU/g Pi county broad bean paste fermented grains Inoculating the inoculated amount of the strain into Pi county broad bean paste to be fermented, fermenting for 7 days, and then adding staphylococcus meat to the strain of the broad bean paste not less than 1 multiplied by 10 7 CFU/g Pi county broad bean paste fermented grains Inoculating the inoculated amount into the Pi county broad bean paste fermented grains to be fermented, fermenting for 7 days, and fermenting with candida variabilis at a ratio of 1×10 or more 7 CFU/g Pi county broad bean paste fermented grains Inoculating the seed quantity of the broad bean paste to be fermented into the Pi county broad bean paste to be fermented for continuous fermentation; the number ratio of Pediococcus acidilactici, staphylococcus botulinum and Candida variabilis is 1:1:1.
2. The method of claim 1, wherein the pediococcus acidilactici, the staphylococcus botulinum and the candida variabilis are cultivated in a synthetic medium to obtain a bacterial liquid, and the bacterial liquid is inoculated into the Pi county broad bean fermented grains to be fermented;
the synthetic medium contains: disodium hydrogen phosphate 6g/L, potassium dihydrogen phosphate 3g/L, glucose 4.5g/L, fructose 1.98g/L, mannitol 0.91g/L, arabinose 1.8g/L, threonine 0.84g/L, isoleucine 0.80.84g/L, leucine 1.38g/L, phenylalanine 0.81g/L, glycine 0.51g/L, alanine 1.06g/L, lysine 1.53g/L, histidine 0.06g/L, arginine 1.73g/L, methionine 0.25g/L, serine 0.15g/L, proline 0.81g/L, valine 0.98g/L, glutamic acid 2.95g/L aspartic acid 1.76g/L, tyrosine 0.85g/L, glutamine 0.51g/L, cysteine 0.02g/L, EDTA mg/L, ferric trichloride hexahydrate 8.3mg/L, zinc chloride 0.84mg/L, cupric chloride dihydrate 0.13mg/L, cobalt chloride hexahydrate 0.1434mg/L, boric acid 0.1mg/L, manganese chloride tetrahydrate 0.016mg/L, ammonium sulfate hydrochloride 1mg/L, riboflavin 1mg/L, niacin 1mg/L, calcium pantothenate 1mg/L, pyridoxine 2mg/L, biotin 10mg/L, folic acid 1mg/L, p-aminobenzoic acid 10mg/L, 1M CaCl 2 1mg/L、1M MgSO 4 0.3mg/L。
3. Use of the method of any one of claims 1-2 for improving the flavor of Pi county bean cotyledon.
4. A use according to claim 3, characterized in that the use includes, but is not limited to, adding the pediococcus acidilactici, staphylococcus sarcoidocus and candida mutans to the fermented soybean paste of p.county beans to be fermented.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949592A (en) * 2018-06-22 2018-12-07 广西顶俏食品有限公司 A kind of microbial immobilized fermentation composition of soy sauce and preparation process and application method
CN111019860A (en) * 2019-12-18 2020-04-17 江南大学 Pediococcus acidilactici for reducing biogenic amine and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108949592A (en) * 2018-06-22 2018-12-07 广西顶俏食品有限公司 A kind of microbial immobilized fermentation composition of soy sauce and preparation process and application method
CN111019860A (en) * 2019-12-18 2020-04-17 江南大学 Pediococcus acidilactici for reducing biogenic amine and application thereof

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