CN111248409A - A kind of low-salt bean paste fermentation method - Google Patents

Abstract

The invention relates to a low-salt thick broad-bean sauce fermentation method, the multi-strain synergistic fermentation method and the application thereof in thick broad-bean sauce provided by the invention realize low salt of products, and the total amount of flavor substances of the prepared low-salt thick broad-bean sauce is respectively increased by 50.4% and 111.1% compared with the total amount of flavor substances of the thick broad-bean sauce prepared under medium-concentration and high-concentration salt concentrations, so that after the fermentation is finished, the sauce mash with 6% of salt content has fuller flavor and mellow fragrance; the organic acid content is also increased by 14.7% and 27.4% compared with the thick broad-bean paste prepared under medium concentration and high concentration salt concentration, so that the taste is better and softer, and the unique flavor is formed. Effectively improves the flavor and the quality of the soybean paste, and further guides the upgrading and updating of the traditional soybean paste industry.

Description

A kind of low-salt bean paste fermentation method

technical field

The invention relates to a low-salt bean paste fermentation method, belonging to the technical field of bioengineering fermentation .

Background technique

Doubanjiang is a traditional fermented condiment in my country. It is usually made from broad beans, flour, pepper and salt as the main raw materials, and is fermented by various microorganisms such as Aspergillus, yeast and bacteria. The production of traditional bean paste is an open process of multi-strain mixed solid-state fermentation, which mainly includes two parts. The first is to inoculate pure Aspergillus oryzae after pretreatment of raw materials for 2-3 days of ventilation koji-making. Then it is natural fermentation, the finished koji is fermented again after mixing with salt water in a large ceramic vat, sun exposure and night dew for 3 to 5 months, and artificially fermenting grains during fermentation is the main step in the production process of bean paste. During the long fermentation process, the protein, starch, fat and other components in broad beans and flour are decomposed into small molecules such as sugar and amino acids under the action of various enzymes secreted by Aspergillus oryzae, and then bacteria and yeast use these small molecules to further synthesize various flavor compounds that ultimately form the sauce's unique flavor.

Salt is the main component in the fermentation process of broad bean paste. In the open-air fermentation environment, salt plays an important role in screening salt-tolerant functional flora and inhibiting the growth of spoilage bacteria and pathogenic bacteria. In addition, recent studies have found that a high-salt diet is directly related to the induction of high blood pressure, heart disease, kidney disease, and cerebral hemorrhage. Therefore, the low-saltization of soy fermented food has attracted extensive attention from all walks of life. However, in the actual production process, the reduction of salinity can easily lead to the growth and reproduction of harmful microorganisms, resulting in the peculiar smell of fermented grains. There are also reports of fermentation under aseptic conditions, but due to technical limitations, the structure and function of functional microorganisms in the natural fermentation state have not been thoroughly analyzed, so it is impossible to achieve better fermentation effects in aseptic culture conditions, such as Some companies have used a single pure Aspergillus oryzae to industrially produce soybean paste, but its flavor and taste are not as rich as traditional fermented soybean paste. Therefore, it is imperative to use a defined starting starter in a sterile environment.

SUMMARY OF THE INVENTION

In view of the above problems, the present invention provides a multi-strain synergistic low-salt bean paste fermentation method, the salinity of the fermented grains is controlled at 6-9%, the bean paste can be fully fermented, the product is low in salinity, and effective to improve the flavor and quality of bean paste.

The invention provides a low-salt bean paste fermentation method, which comprises adding yeast combined with luteolin, Staphylococcus meatus, Bacillus subtilis and Lactobacillus weissi for fermentation during the fermentation process of soybean paste.

In one embodiment of the present invention, the combined yeast is Zygosaccharomyces rouxii Y-8, which has been deposited by the General Microbiology Center of the China Microorganism Culture Collection on July 25, 2019, The deposit number is CMCC No.18293, and the deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the Staphylococcus carnosus is Staphylococcus carnosus M43, in July 2019 On the 25th, it was preserved by the General Microbiology Center of the China Microorganism Culture Collection Management Committee, the preservation number is CGMCC No.18295, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the Bacillus subtilis (Bacillus subtilis) is disclosed in the publication number: CN106867938B, the invention title is "a strain of Bacillus subtilis that reduces biogenic amines and its application" in the patent document; the fusion Lactobacillus Weissella (Weissella confusa) is disclosed in the publication number It is in the patent document of CN103571782B "A Fusion Lactobacillus and its Application".

In one embodiment of the present invention, the specific steps of the fermentation method are: inserting Aspergillus oryzae into a mixed system of broad bean and wheat flour for culturing to prepare koji; mixing the koji and salt water to make fermented grains; Inoculated with Staphylococcus meatus, fused Lactobacillus weissii, Bacillus subtilis and combined yeast for fermentation; after the fermentation is completed, fava bean paste is prepared.

In an embodiment of the present invention, the mass ratio of the broad bean to the wheat flour is 2-6:1-2.

In an embodiment of the present invention, the koji is prepared after culturing at 25-35° C. for 50-70 hours.

In an embodiment of the present invention, the inoculum amount of the Aspergillus oryzae is a final concentration of 1×10 ⁵ to 1×10 ⁷ spores/g.

In an embodiment of the present invention, the inoculum amounts of the Staphylococcus caryotis, Bacillus subtilis, Lactobacillus fusiformis, and Yeast combined routii are the final concentrations of 1×10 ⁹ to 1×10 ¹⁰ cfu/g, 1×10 ⁹ to 1×10 ¹⁰ cfu/g, 1×10 ⁸ to 1×10 ⁹ cfu/g, and 10 ⁴ to 10 ⁵ cfu/g.

In an embodiment of the present invention, the final concentration of the brine in the fermentation system is 5 g/L to 15 g/L.

In an embodiment of the present invention, the fermentation time is 5-8 weeks.

The present invention provides a preparation method of soybean paste with increased flavor. The method is to add combined yeast, Staphylococcus meatus, Bacillus subtilis and Lactobacillus weissi to ferment the soybean paste before fermentation.

The present invention provides a method for rapidly preparing bean paste. The method is to add combined yeast, Staphylococcus meatus, Bacillus subtilis, and Lactobacillus weissii to ferment the soybean paste before fermentation.

The present invention provides a starter, the starter comprising Yeast rutheli, Staphylococcus meatus, Bacillus subtilis, and Lactobacillus weissiferans.

The invention provides a preparation method of a starter. The method comprises the following steps: inoculating Staphylococcus carves, Bacillus subtilis, Lactobacillus weissii, and combined yeasts into a culture medium, and activating at 30-40° C. In the middle and late logarithmic stage, subculture for 2 to 3 times until the cell concentration in the bacterial liquid reaches more than 10 ⁶ cfu/mL of viable cells, centrifuge the bacterial liquid at 6000 to 10000 rpm for 15 to 25 min to collect the precipitate, and add it to the precipitate in turn. Buffer and cryoprotectant, when the cell concentration is not less than 10 ⁷ cfu/mL, vacuum freeze-drying is carried out to obtain freeze-dried powders of different strains respectively; , and Roux combined yeast powder in the ratio of (1× ¹⁰⁵ ～1× ¹⁰⁶ ):(1× ¹⁰⁵ ～1× ¹⁰⁶ ):(1× ¹⁰⁴ ～1× ¹⁰⁵ ):1 to obtain the starter .

In one embodiment of the present invention, when the cell concentration is not less than 10 ⁸ cfu/mL, vacuum freeze-drying is performed.

In one embodiment of the present invention, the buffer is double distilled water or phosphate buffer solution, and the cryoprotectant is trehalose or skim milk powder.

In one embodiment of the present invention, the buffer is 0.1-1M phosphate buffer with pH value of 5-8, and the cryoprotectant is 5%-20% (w/v) trehalose and / or skim milk powder.

In one embodiment of the present invention, the buffer is 0.2M phosphate buffer with pH 7, and the cryoprotectant is 10-15% (w/v) trehalose or skim milk powder.

In one embodiment of the present invention, Staphylococcus meatus, Bacillus subtilis, Lactobacillus weissii, and combined yeast powders are prepared in the form of (1×10 ⁴ to 1×10 ⁵ ): (1×10 ⁴ to 1 ×10 ⁵ ): (1 × 10 ³ -1 × 10 ⁴ ): 1 by mixing to obtain a starter.

In one embodiment of the present invention, Staphylococcus caris, Bacillus subtilis, Lactobacillus weissii, and combined yeast powder are mixed in a ratio of 1×10 ⁵ : 1×10 ⁵ : 1×10 ⁴ : 1 Get a starter.

The present invention provides the application of a starter in the preparation of fermented soybean food, and the food includes broad bean paste, soybean paste and soy sauce.

The invention provides the application of a low-salt bean paste fermentation method in the preparation of bean fermented food, and the food includes broad bean paste, soybean paste and soy sauce.

Beneficial effects of the present invention: the multi-strain synergistic fermentation method provided by the present invention and its application in the bean paste realize the low salinity of the product, and can well ferment the bean paste at a salinity of 6%. The obtained low-salt bean paste has an increase of 50.4% and 111.1% in the total amount of flavor substances compared with the bean paste prepared under the medium concentration and high salt concentration, respectively, so that after the fermentation, the flavor of the fermented grains with 6% salinity is more plump. The aroma is more mellow; the organic acid content is also increased by 14.7% and 27.4% compared with the bean paste prepared under the medium concentration and high concentration salt concentration, which makes the taste better and softer, thus forming a unique flavor.

biological material preservation

Zygosaccharomyces rouxii, has been deposited by the General Microbiology Center of the China Microorganism Culture Collection on July 25, 2019, the deposit number is CMCC No. 18293, and the deposit address is No. 1 Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences.

Staphylococcus carnosus has been preserved by the General Microbiology Center of the China Microbial Culture Collection and Administration Commission on July 25, 2019, the preservation number is CGMCC No.18295, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing. 3, Institute of Microbiology, Chinese Academy of Sciences.

Description of drawings

Figure 1 is the metabolic profile of the strain. CK: control; A: Aspergillus oryzae; B: Bacillus subtilis; S: Staphylococcus meatus; W: Weisseria fused; Z: Aspergillus ruberii. (a) enzymatic activity; (b) volatile flavor substances; (c) non-volatile organic acids; (d) free amino acids.

Fig. 2 is the OD ₆₀₀ values of Aspergillus oryzae, Yeast ruberii, Bacillus subtilis, Lactobacillus fusiformis and Staphylococcus meatus cultured in liquid medium with different salinity for 72 hours.

Fig. 3 is the bacteriostatic effect of Aspergillus oryzae, Yeast rutheli, Bacillus subtilis, Lactobacillus fusiformis and Staphylococcus meatus on common harmful bacteria in the fermentation process of soy fermented grains. A: Aspergillus oryzae; B: Bacillus subtilis; S: Staphylococcus meatus; W: Weisseria fusiformis; Z: Aspergillus ruberii.

Fig. 4 is the biomass of the single and double combination culture and fermentation of Aspergillus oryzae, combined yeast, Bacillus subtilis, Lactobacillus fusiformis and Staphylococcus meatus strains. A: Aspergillus oryzae; B: Bacillus subtilis; S: Staphylococcus meatus; W: Weisseria fused;

Fig. 5 is the fermentation characteristics of doubanjiang under different salinity conditions of multi-strain co-fermentation; (a) amino acid nitrogen, (b) organic acid, (c) non-volatile organic acid, (d) volatile flavor substance; Wherein, L is the fermented fermented grain obtained by inoculating Aspergillus oryzae, combined yeast, Bacillus subtilis, Lactobacillus weissii and Staphylococcus meatus when the salinity is 6%, and M is the inoculation of Aspergillus oryzae, Lulu The fermented fermented grains obtained by the fermentation of combined yeast, Bacillus subtilis, Lactobacillus fusiformis and Staphylococcus meatus, H is inoculated with Aspergillus oryzae, combined yeast rutheli, Bacillus subtilis, Lactobacillus fusiformis when the salinity is 12% The fermented fermented grains obtained by fermenting with Staphylococcus meatus, A is the bean paste fermented grains inoculated with Aspergillus oryzae only when the salinity is 12%.

Detailed ways

The Bacillus subtilis used in the present invention is shown in the patent "A Bacillus subtilis that reduces biogenic amines and its application" (publication number: CN106867938B); the fusion Lactobacillus (Weissella confusa) is shown in the patent "A Strain Fusion Lactobacillus and its Application" (publication number: CN103571782B); Aspergillus oryzae 3.042 was purchased from commercial channels. Example 1 Analysis of traditional bean paste microbial community

The microbial community structure during the fermentation process of traditional bean paste was analyzed by amplicon sequencing, and it was found that the dominant bacterial genera in the soybean paste were Staphylococcus, Bacillus and Weissella. The genus Weissella accounted for 79.8%, 7.5% and 5.9% of the total bacteria, respectively; the fungal genera were mainly Aspergillus and Zygosaccharomyces, accounting for 97% and 1.3% of the total fungi, respectively. Then, the microorganisms in the soy fermented grains were isolated and purified and based on the food safety of the strains, the representative species of the five dominant microbial genera were determined, namely Staphylococcus carnosus, Bacillus subtilis, fusion Weissella confusa, Aspergillus oryzae and Zygosaccharmyces rouxii.

The cultivation of the bacterial strain of embodiment 2

PDA culture medium: 200 g of potato is added with water to cook and filter, add 20 g of glucose, 20 g of agar, and dilute to 1L with deionized water.

YPD medium: 20 g of glucose, 20 g of peptone, 10 g of yeast powder, and make up to 1 L with deionized water.

MRS medium: glucose 20g, peptone 10g, beef extract 10g, yeast powder 5g, sodium acetate 5g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, Tween-80 1.0mL, magnesium sulfate 0.58g, manganese sulfate 0.25g g, make up to 1L with deionized water.

Aspergillus oryzae was inoculated on PDA slant medium, cultured at 28°C for 2-3 days, and eluted with 0.9% saline to prepare a spore suspension.

Inoculated in MYPD liquid medium with 1% inoculum of Yeast ruberii, and shaken at 30°C and 200rpm to culture to the middle and late logarithmic growth period, and the OD ₆₀₀ was 0.8-1.0.

Staphylococcus caryotis and Lactobacillus fusiformis were inoculated into MRS liquid medium at an inoculum of 1%, and cultured with shaking at 37° C. and 200 rpm to the middle and late stage of logarithmic growth, with an OD ₆₀₀ of 0.8-1.0.

Bacillus subtilis was inoculated into LB liquid medium with 1% inoculum, and cultured with shaking at 37°C and 200 rpm to the middle and late stage of logarithmic growth, with an OD ₆₀₀ of 0.8-1.0.

The functional verification experiment of the strain of embodiment 3

The functions of Aspergillus oryzae, combined yeast, Staphylococcus meatus, Bacillus subtilis and Lactobacillus fusiformis were verified by in vitro mixed culture fermentation method.

Add 10 g of broad bean flour, 2.5 g of wheat flour, and 50 g of broad bean paste to 1.0 L of deionized water to cook for 30 min, and filter to prepare a simulated liquid medium. Then these strains were inoculated into liquid medium to simulate the fermentation process of bean paste, and the initial cell density of each strain was adjusted to 1.0×10 ⁶ CFU/g. All fermentations were carried out at 30 °C for 3 days. After the fermentation, the flavor substances of the fermentation broth of single bacteria were detected, and the uninoculated fermentation medium was used as the control. All experiments were performed in triplicate.

Doubanjiang organic acid content determination method: weigh 2g-10g of fermented grains and put into 50mL EP tube, add 40mL ultrapure water and soak for 2h, shake once every 15min, and centrifuge at 7500rpm for 5min. Take 1 mL of the supernatant into a 2 mL EP tube, add 0.4 mL each of 106g/L potassium ferrocyanide solution and 0.4 mL of zinc sulfate solution to remove protein and pigment, settle for 2 h (vortexing), and then centrifuge at 7500 rpm for 3 min, take the supernatant Centrifuge at 10,000 rpm for 5 min, and then pass through a 0.22 μm filter (green water system).

Organic acid standard samples: oxalic acid, pyruvic acid, acetic acid, lactic acid, and succinic acid with concentrations of 0.05, 0.05, 0.50, 0.50, 0.50, and 1.00 mg/mL, respectively.

Analysis conditions: Chromatographic column: Waters Atlantis T3 (5um, 4.6×250mm); column temperature: 40°C; mobile phase: accurately weigh 3.12g of NaH2PO4 2H2O, dilute to 1000mL with deionized water, and adjust the pH of the solution with H3PO4 to 2.7; injection volume: 10uL; flow rate: 0.7mL/min; detection wavelength: UV210 nm.

As shown in Figure 1, Aspergillus oryzae and Bacillus subtilis are the main microorganisms that secrete protease and starch glucoamylase. Amylase was 178.0U/g and 24.5U/g, respectively, while the protease and amylase secreted by Gluconococcus maculatum were only 30.7U/g and 30.0U/g, respectively. Amyloamylase was only 21.2U/g and 20.7U/g respectively, and neither could secrete protease; therefore, Aspergillus oryzae and Bacillus subtilis secrete protease and amyloamylase, which may play a certain role in the degradation of macromolecular substances.

In addition, in the medium inoculated with combined yeast, the contents of esters and alcohols increased significantly, which were 906.3 μg/L and 1738.2 μg/L, respectively, which were 68.9 times and 15.5 times higher than that of the control, and higher than that of Aspergillus oryzae. It was increased by 17.2 and 9.6%, respectively, 16.6 times and 14.2 times higher than that of Bacillus subtilis, 26.8 times and 10.3 times higher than that of Gluconococcus caris, and 16.6 times and 3.8 times higher than that of fusion Lactobacillus weissus, respectively. We also found that Aspergillus oryzae and Staphylococcus meatus were able to synthesize acetic acid (4.6g/L and 3.8g/L, respectively), while only 1.8g/L and 2.3g/L in control, Bacillus subtilis, and Junctional yeast, respectively and 1.3g/L; while the fusion of Lactobacillus weissiferans can not only synthesize acetic acid (3.7g/L), but also synthesize high content of lactic acid (4.7g/L), while Aspergillus oryzae, Staphylococcus meatus, Bacillus subtilis, Only 2.0g/L, 1.0g/L, 1.8g/L, respectively, in Staphylococcus caryotis and combined yeast. This indicated that Lactobacillus weissus, Aspergillus oryzae and Staphylococcus caryopsis had higher organic acid production capacity, and these organic acids further led to the increase of titratable acid and the decrease of pH value during the fermentation of faba bean paste.

Similar to the synthesis ability of organic acids, the fusion Lactobacillus oryzae, Aspergillus oryzae and Staphylococcus meat also have higher amino acid synthesis ability or protein degradation ability, and the free amino acid content is 26.0g/kg, 23.2g/kg, 23.1g/kg, respectively. g/kg, while the free amino acid contents of the control, Bacillus subtilis, and combined yeasts were 20.4 g/kg, 16.2 g/kg, and 20.4 g/kg, respectively.

Embodiment 4 Strain salinity tolerance experiment

To prepare media with different salt concentrations: 0%, 4%, 8%, 12%, 16% and 20% (w/v) NaCl were added to MYPD, MRS or LB liquid media, respectively. Aspergillus oryzae, combined yeast, staphylococcus meatus, Bacillus subtilis and Lactobacillus fusiformis cultivated to the middle and late logarithmic growth in Example 1 were inoculated into corresponding PDA (Aspergillus oryzae), MYPD (Ruchnerii), respectively. Combine yeast), MRS (Staphylococcus meatus and Lactobacillus fusiformis) or LB (Bacillus subtilis) liquid medium so that its final concentration in the medium is 10 ⁶ CFU or spores/g after inoculation and cultured for 3 Days later, the growth status of each strain under different salinity conditions was determined, and the cell concentration was calculated from the absorbance value at 600 nm to evaluate its salt tolerance.

As shown in Figure 2, Aspergillus oryzae, Bacillus ruberii, Staphylococcus molluscs, Bacillus subtilis, and Lactobacillus weissii can all grow on the medium containing 8% NaCl, among which Aspergillus oryzae, Bacillus subtilis and Weiss Lactobacillus formulae was less salt tolerant, barely growing at 12% salinity, while S. rutheli and S. carnivora showed good salt-tolerant growth. The growth rate of combined yeast and Staphylococcus meatus was faster under the conditions of NaCl final concentration of 4% and 8%, followed by the growth rate of NaCl final concentration of 0%, 12% and 16%, and the final NaCl concentration of 20%. At %, the growth rate of J. rutheli strains was relatively slow, while Staphylococcus meatus could still grow.

Example 5 Bacteriostatic test of bacterial strains

The filter paper method was used to conduct the antibacterial test on the common harmful bacteria in the fermentation process of soy fermented grains. First, spread 100 μL of indicator bacteria growing to the log phase on the plate (Bacillus cereus, Staphylococcus aureus and Escherichia coli screened from the soy fermented grains in the laboratory in the preliminary test were selected as indicator bacteria), and then 6 mm The round sterile filter paper is evenly dipped in the culture solution of the experimental bacteria and placed flat in the middle of the agar plate. Sterile water was used as a negative control, and after incubation at 30 °C for 48 h, the growth inhibition was evaluated by the size of the inhibition zone. As shown in Figure 3, Bacillus subtilis can inhibit the growth of Bacillus cereus; fusion of Lactobacillus weissiformis can inhibit the growth of Escherichia coli; Staphylococcus caryotis and Weisseria species can inhibit the growth of Staphylococcus aureus.

Example 6 Interaction experiments of strains

Microbial interactions among Aspergillus oryzae, Yeast rutheli, Staphylococcus meatus, Bacillus subtilis, and Lactobacillus weissiferans were analyzed using an in vitro mixed culture fermentation method.

Add 10 g of broad bean flour, 2.5 g of wheat flour, and 50 g of broad bean paste to 1.0 L of deionized water to cook for 30 min, and filter to prepare a simulated liquid medium. These strains were then inoculated into liquid medium to simulate the fermentation process of bean paste, and the initial cell density of each strain was adjusted to 1.0×10 ⁶ CFU or spore/g. All fermentations were carried out at 30°C for 3 days. The uninoculated fermentation medium was used as the control. The cells of different strains were counted by real-time fluorescence quantitative PCR technology after fermentation. All experiments were performed in triplicate. Microbial interactions between different species can affect their growth, which in turn affects the overall microbial community structure and its metabolic activities. As shown in Figure 4, Aspergillus oryzae (A) and Bacillus subtilis (B) inhibited each other, and the number of cells both decreased under co-culture conditions. Similar competition exists between Bacillus subtilis (B) and Lactobacillus fusiformis (W). In addition, except for the fusion of Lactobacillus weissus (W), which has a significant inhibitory effect on Staphylococcus cariosus (S), the other strains have no significant effect on Staphylococcus cariosus. Bacillus subtilis has a certain inhibitory effect on most microorganisms. In addition, a significant reciprocity was found between fused L. weissii and Y. ruberii. This interaction between lactic acid bacteria and yeast is also prevalent in fermented foods.

Example 7 Application of multi-strain synergistic fermentation in soybean paste fermentation

Experimental analysis shows that although Bacillus subtilis can inhibit the growth of Aspergillus oryzae, it has a certain ability to secrete enzymes. Therefore, it can release various enzymes and hydrolyze proteins and starches together with Aspergillus oryzae in the early stage of fermentation. growth provides precursors. In addition, Aspergillus oryzae, Staphylococcus meatus and Lactobacillus fusiformis have high organic acid and amino acid synthesis ability, which can lead to acidification of soybean paste and provide unique flavor for soybean paste. As the fermentation progresses, Aspergillus oryzae, Bacillus subtilis and Lactobacillus weissii gradually die out in the middle and late stages of fermentation due to their low salt tolerance. On the other hand, with the release of the inhibition of Bacillus subtilis on binding yeast, and the fusion of Lactobacillus weissii and Staphylococcus meatus in the early stage created a lower pH environment for the growth of binding yeast, the salt-resistant binding Yeast began to play a role, mainly responsible for the formation of various volatile flavor components such as alcohols and esters, which directly led to the unique flavor of bean paste. Due to the higher salt tolerance of S. carnosus and Y. rutheli, they play an important role in the middle and later stages of fermentation.

The fermentation performance of the synthetic community at different salinities was compared by inoculating Aspergillus oryzae, combined yeast, Staphylococcus meatus, Bacillus subtilis and Lactobacillus fusogenis during the fermentation process.

Broad beans were denatured by high-pressure cooking at 121°C, cooled to room temperature, fully mixed with wheat flour (3:1, w/w), and inoculated with Aspergillus oryzae with a final concentration of 10 ⁶ spores/g, and cultured at 30°C for 60 hours. song. The koji was mixed with different concentrations of brine to a salt concentration of 6%, 9%, and 12%, respectively, with a final moisture content of 55%. Inoculate Staphylococcus meatus, Lactobacillus fusiformis, Bacillus subtilis, and combined yeasts of Lloyd's yeast activated to the middle and late logarithm into the fermented glutinous rice flour, so that the final concentrations in the fermented glutinous rice flour are 10 ⁹ cfu/g, 10 9 cfu/g, 10 ⁹ cfu/g, 10 ⁸ cfu/g, 10 ⁴ cfu/g, with the uninoculated fermented grains as the control, the fermentation temperature was 30℃, and the amino acid nitrogen in the bean paste prepared under different salt concentrations was tracked and determined content and titratable acid content (see GB/T 5009.40-2003 for specific steps). It was found that the amino acid nitrogen content of all groups reached a steady state after about 4 weeks, indicating that the fermentation was basically over (Table 1).

Table 1 Amino acid nitrogen (%) in bean paste samples with different salt concentrations

Note: L is the soy fermented grain fermented by inoculation with Aspergillus oryzae, combined yeast, Bacillus subtilis, Lactobacillus weissii and Staphylococcus meatus when the salinity is 6%, M is the inoculation of Aspergillus oryzae, Lulu The fermented fermented grains obtained by the fermentation of combined yeast, Bacillus subtilis, Lactobacillus fusiformis and Staphylococcus meatus, H is inoculated with Aspergillus oryzae, combined yeast rutheli, Bacillus subtilis, Lactobacillus fusiformis when the salinity is 12% The fermented fermented grains obtained by fermenting with Staphylococcus meatus, A is the bean paste fermented grains inoculated with Aspergillus oryzae only when the salinity is 12%.

The fermentation performance of synthetic microbial communities under different salinity fermentation conditions was compared. As shown in Figure 5, the lower the salinity, the higher the growth rate and content of organic acids in the synthetic community (Table 2, see Example 3 for the organic acid determination steps). The total amount can reach 140.71g/kg, which is 1.5 times higher than that of the control with only Aspergillus oryzae added, and the organic acid content of bean paste prepared with medium and high salt concentrations is increased by 14.7% and 27.4% respectively; and flavor substances The higher the content of salt, the total amount of flavor substances in the doubanjiang prepared under the medium and high salt concentrations increased by 50.4% and 111.1%, respectively. After fermentation, the fermented grains with 6% salinity have a fuller flavor and a more mellow aroma. This shows that low salt can effectively promote the efficient fermentation of bean paste and improve the flavor of broad bean paste (Table 3).

Determination method of bean paste flavor substances: accurately weigh about 2g of soy fermented grains into a 15ml solid phase microextraction bottle, add 2g solid NaCl, 6mL decarbonated water respectively, and add 20μL 25mg/L 2-octanol internal standard, put After entering the rotor, tighten the lid and put it into a constant temperature water bath with a magnetic stirrer at 55°C, and the magnetic stirring speed is 400 r/min. Insert the extraction head into the air part of the bottle through the solid phase microextraction bottle cap, push out the fiber head, turn on the magnetic stirrer, and withdraw the fiber head after headspace adsorption for 40 minutes.

Chromatographic conditions:

DB-Wax capillary column, column length 30mm, inner diameter 0.32mm, liquid film thickness 0.25μm; carrier gas: He; flow rate: 10mL/min, splitless; column temperature: the temperature of the injection port is kept at 250°C; the heating program is set As follows: the initial gas chromatographic column temperature is 40 °C, maintained for 3.5 min, heated to 90 °C at 5 °C/min, and then heated to 230 °C at 12 °C/min, maintained for 10 min.

MS conditions:

Ionization mode: EI+; emission current (μA): 200; drive voltage (V): 0.5; electron energy (eV): 70; lens 1 (V): 4, lens 2 (V): 35; ion energy (eV) : 1.8; Ion energy ramp (mV·amu-1): 0.0; Interface temperature (°C): 250; Ion source temperature (°C): 200; Low mass resolution: 18.0; Mass resolution: 12.6; Detector voltage (V): 350; scan mass range: 33-450 amu.

The organic acid content (g/kg) in table 2 different salt concentration bean paste samples

Note: L is the soy fermented grain fermented by inoculation with Aspergillus oryzae, combined yeast, Bacillus subtilis, Lactobacillus weissii and Staphylococcus meatus when the salinity is 6%, M is the inoculation of Aspergillus oryzae, Lulu The fermented fermented grains obtained by the fermentation of combined yeast, Bacillus subtilis, Lactobacillus fusiformis and Staphylococcus meatus, H is inoculated with Aspergillus oryzae, combined yeast rutheli, Bacillus subtilis, Lactobacillus fusiformis when the salinity is 12% The fermented fermented grains obtained by fermenting with Staphylococcus meatus, A is the bean paste fermented grains inoculated with Aspergillus oryzae only when the salinity is 12%.

Table 3 Contents of various volatile substances (mg/kg) in bean paste samples with different salt concentrations

Note: L is the soy fermented grain fermented by inoculation with Aspergillus oryzae, combined yeast, Bacillus subtilis, Lactobacillus weissii and Staphylococcus meatus when the salinity is 6%, M is the inoculation of Aspergillus oryzae, Lulu The fermented fermented grains obtained by the fermentation of combined yeast, Bacillus subtilis, Lactobacillus fusiformis and Staphylococcus meatus, H is inoculated with Aspergillus oryzae, combined yeast rutheli, Bacillus subtilis, Lactobacillus fusiformis when the salinity is 12% The fermented fermented grains obtained by fermenting with Staphylococcus meatus, A is the bean paste fermented grains inoculated with Aspergillus oryzae only when the salinity is 12%.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (10)

1. a low-salt bean paste fermentation method, is characterized in that, described method is to add Staphylococcus meatus, Bacillus subtilis, fused Lactobacillus weissii and combined yeast of Rou Shi at the fermented stage of fermented soybean paste; The ratio of cocci, Bacillus subtilis, Lactobacillus fusiformis and Yeast routii is (1×10 ⁴ ～1×10 ⁶ ):(1×10 ⁴ ～1×10 ⁶ ):(1×10 ³ ～ 1×10 ⁵ ): 1; the fermented fermented soybean paste was fermented in a sterile environment. 2. method according to claim 1, is characterized in that, described combined yeast is combined yeast (Zygosaccharomyces rouxii) Y-8, has been on July 25, 2019 by China Microorganism Culture Collection Management Committee Preserved by the General Microbiology Center, the preservation number is CMCC No. 18293; the Staphylococcus carnosus is Staphylococcus carnosus M43, which was preserved by the General Microbiology Center of the China Microorganism Culture Collection on July 25, 2019, and the preservation number is It is CGMCC No.18295. 3. method according to claim 1, is characterized in that, described method is to connect Aspergillus oryzae in the mixed system of broad bean and wheat flour to cultivate and prepare koji; koji and salt water are mixed to make soy glutinous rice grains; The fermented grains are inoculated with Staphylococcus meatus, fused Lactobacillus weissii, Bacillus subtilis and combined yeast for fermentation to prepare bean paste. 4 . The method according to claim 3 , wherein the inoculum amount of the Staphylococcus meatus, Bacillus subtilis, Lactobacillus fusiformis, and combined yeasts is a final concentration of 1×10 ⁸ to 1×10 . 5 . ¹⁰ cfu/g, 1×10 ⁸ to 1×10 ¹⁰ cfu/g, 1×10 ⁷ to 1×10 ⁹ cfu/g, and 1×10 ³ to 1×10 ⁵ cfu/g. 5 . The method according to claim 3 , wherein the final concentration of the brine in the fermentation system is 5 g/L to 20 g/L. 6 . 6 . The method according to claim 3 , wherein the fermentation time is 5-8 weeks. 7 . 7. A method for improving the flavor of bean paste, characterized in that the method is to ferment by adding combined yeast, Staphylococcus meatus, Bacillus subtilis, and Lactobacillus weissii before fermentation of soybean paste. 8. A starter, characterized in that, the starter comprises Yeast rutheli, Staphylococcus caryotis, Bacillus subtilis, and Lactobacillus weissii. 9 . The starter according to claim 8 , wherein the starter has a ratio of Staphylococcus meatus, Bacillus subtilis, Lactobacillus fusiformis, and combined yeasts of (1×10 ⁴ -1 ×10 ⁶ ):(1×10 ⁴ to 1×10 ⁶ ):(1×10 ³ to 1×10 ⁵ ):1. 10. The application of any one of the methods of claims 1 or 3 to 7 or the starter of claims 8 or 9 in the preparation of fermented soy foods including broad bean paste, soybean paste and soy sauce.

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