CN117050897A - Composite starter and application thereof in low-salt fermentation of peppers - Google Patents

Abstract

The invention discloses a composite leavening agent, which comprises the following components: lactobacillus plantarum bacterial liquidLactobacillus plantarumMeng Maiye Saccharomyces cerevisiae liquidMeyerozyma guilliermondiiThe method comprises the steps of carrying out a first treatment on the surface of the The lactobacillus plantarumLactobacillus plantarumThe preservation number is CGMCC NO.27361, and the preservation time is 2023, 5 and 17 days; the Meng Maiye yeastMeyerozyma guilliermondiiThe preservation number is CGMCC No.27360, and the preservation time is 2023, 5 and 17 days. The composite starter of the invention is preparedAfter the low-salt fermented chilli is prepared, sensory evaluation, determination of pH, total acid, reducing sugar and amino acid nitrogen are carried out, so that compared with other groups, the low-salt fermented chilli is faster in fermentation, more in acid production and best in fermentation aroma quality.

Description

Composite starter and application thereof in low-salt fermentation of peppers

Technical Field

The invention belongs to the technical field of food fermentation, and particularly relates to a composite starter and application thereof in low-salt fermentation of capsicum.

Background

Flavor substances in the fermented peppers: organic acid, capsaicin, amino acid nitrogen, sugar, salt and the like, and shows five tastes of acid, hot, fresh, sweet and salty. Inoculating different strains plays a decisive role in the formation of flavor substances in the fermented capsicum. The fermentation sugar like fermentation bacteria such as streptococcus lactis, lactobacillus plantarum and the like can produce lactic acid, and the fermentation sugar such as heterofermentation bacteria such as leuconostoc mesenteroides, lactobacillus brevis, lactobacillus fermentum and the like can produce ethanol and acetic acid, so that the accumulation of organic acid is influenced; pseudomonas in fermented peppers is closely related to most aldehydes, especially 3-methylbutyraldehyde, 2-methylbutyraldehyde, phenylacetaldehyde, 2-acetylpyrrole, 5-methylfurfural and 5-methyl-2-phenyl-2-hexenal. However, it has been found that inoculation with lactobacillus plantarum reduces the microbial diversity in the fermented capsicum, resulting in a negative correlation with most volatile compounds, resulting in a single flavor of the fermented capsicum product. At present, the chilli products are mainly concentrated on traditional manual making or industrialized flavor blending and production of high-salt fermented chilli (micro-fermented chilli), and a large amount of salted chilli water discharged after the high-salt chilli is processed and desalted not only pollutes the environment, but also causes the loss of chilli flavor and nutrient substances, and the flavor is required to be blended again after fermentation.

Disclosure of Invention

The invention aims to provide a composite starter and application thereof in low-salt fermentation of capsicum, and the application of the composite starter obviously improves the variety and content of volatile flavor substances of fermented capsicum, so that the inoculated fermented capsicum has strong flavor and good quality.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

a composite starter comprising: lactobacillus plantarum bacterial liquid Lactobacillus plantarum and saccharomyces cerevisiae Meyerozyma guilliermondii of Meng Maiye in season; the lactobacillus plantarum Lactobacillus plantarum is preserved in China general microbiological culture collection center (CGMCC), the preservation number is CGMCC NO.27361, the preservation time is 2023, 5 and 17 days, and the preservation address is North Chen Xiyu No. 1, 3 in the Chaoyang area of Beijing city of China;

the Meng Maiye yeast Meyerozyma guilliermondii is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.27360, the preservation time is 2023, 5 and 17 days, and the preservation address is North Chen Xiyu No. 1, 3 in the Chaoyang area of Beijing city.

According to the embodiment of the invention, the preparation method of the lactobacillus plantarum bacterial liquid comprises the following steps: picking single colony to culture medium, culturing at 37+ -5deg.C for 20-28 hr, and activating for several times; and (3) centrifuging, collecting thalli, flushing with sterile physiological saline, and adjusting the concentration of bacterial liquid by using a Maillard turbidimetry method.

The preparation method of the Meng Maiye saccharomyces cerevisiae liquid comprises the following steps: picking single colony to culture medium, culturing at 28+ -5deg.C for 20-28 hr, activating for several times, centrifuging, collecting thallus, washing with sterile physiological saline, and regulating Meng Maiye yeast amount by blood cell counting method.

In one preferred embodiment, the viable count of Lactobacillus plantarum in the Lactobacillus plantarum solution is 1×10 ⁹ ～3×10 ⁹ cfu/mL。

In one preferred embodiment, the number of live Monilinia is 1×10 in the Meng Maiye Saccharomyces cerevisiae solution ⁸ ～3×10 ⁸ cfu/mL。

In a further aspect of the invention, the invention also provides the use of the composite starter in the preparation of fermented peppers.

In yet another aspect of the present invention, the present invention also provides a fermented capsicum. According to an embodiment of the present invention, the fermented capsicum includes: lactobacillus plantarum and Meng Maiye s. The fermented chilli according to the embodiment of the invention has strong flavor and good quality.

In yet another aspect of the present invention, a method of preparing fermented peppers is also provided. According to an embodiment of the present invention, the method includes: the capsicum is fermented using the composite starter as described above, so as to obtain a fermented capsicum. The fermented chilli prepared by the embodiment of the invention has rich flavor and good quality.

According to the embodiment of the invention, the concentration of the fermenting agent is 6-9%, and the strain ratio is 1 (1-2).

According to an embodiment of the invention, the fermentation is a light-protected fermentation at 35℃for 15-20 days.

According to the embodiment of the invention, the chilli contains salt, and the content of the salt is 4% -8% (w/w, unit g/g).

Thus, the study separates lactobacillus and Meng Maiye yeast from natural fermented capsicum, and performs acid production, bacteriostasis, gas production, uncut film production, acid resistance and other experiments on the lactobacillus and Meng Maiye yeast, and initially screens out the lactobacillus and Meng Maiye yeast with excellent fermentation performance. Performing 8% (W/W) low-salt inoculation fermentation on the primarily screened lactobacillus and Meng Maiye yeast, performing sensory evaluation on the fermented peppers through compound fermentation of different strains, measuring the changes of the pH value, total acid, reducing sugar, amino acid nitrogen and other contents in the fermentation process, measuring the contents of organic acid and volatile aroma substances after fermentation, researching the aroma quality in the fermentation process, and screening the compound strain with the best fermentation aroma quality. And carrying out molecular biological identification on the composite strain, and carrying out fermentation with different strain concentrations to obtain the optimal fermentation strain concentration.

Compared with the prior art, the invention has the beneficial effects that:

1. The lactobacillus plantarum provided by the embodiment of the invention has the capability of strongly inhibiting the growth of escherichia coli, staphylococcus aureus and salmonella;

2. after the compound starter is used for preparing the low-salt fermented chilli, the pH value, the total acid, the reducing sugar and the amino acid nitrogen of the compound starter are respectively 3.77,0.74g/100g,1.72g/100g and 0.323g/100g through sensory evaluation and measurement, compared with other groups, the compound starter is faster in fermentation and more in acid production. R is R ₃ :J ₁ The highest comprehensive sensory score was 88.80 points. The organic acid reaches 158.50 +/-0.73 g/kg. R is R ₃ :J ₁ 92 kinds of volatile aroma types are detected, and the highest content of esters is 40.77%. R is R ₃ :J ₁ There are 36 aroma substances with relative odor activity values (rOAVs) greater than 0.1. By principal component analysis, hexyl acetate, linalool, phenethyl alcohol, undecanal, n-hexanal and D-limonene are R ₃ :J ₁ The key aroma substances of the (4) are the best in fermentation aroma quality.

Drawings

FIG. 1 shows colony morphology and cell morphology of lactic acid bacteria;

FIG. 2 shows the pH change after 48h of incubation of different lactic acid bacteria;

FIG. 3 shows the inhibitory effect of different lactic acid bacteria fermentation broths on pathogenic bacteria;

FIG. 4 is a description of colony morphology and cell morphology of 7 strains of yeast;

FIG. 5 is a diagram showing the status of different yeasts producing the purity;

FIG. 6 is acid resistance of different yeasts;

FIG. 7 shows the pH change of the low-salt fermented peppers compounded by different strains;

FIG. 8 shows the total acid change of the low-salt fermented peppers compounded by different strains;

FIG. 9 shows the change of reducing sugar of the low-salt fermented peppers compounded by different strains;

FIG. 10 shows the amino acid nitrogen change of the different strain compound low-salt fermented peppers;

FIG. 11 shows the types and contents of volatile aroma substances of different fermented peppers;

FIG. 12 is a volatile aroma cluster heat map;

FIG. 13 shows the electrophoresis pattern of the R3 PCR products of lactic acid bacteria (a) and phylogenetic tree (b);

FIG. 14 shows the electrophoresis pattern of the PCR products of yeast J1 (a) and phylogenetic tree (b);

FIG. 15 shows pH variation of low salt fermented peppers with different bacteria concentrations;

FIG. 16 shows the total acid change of low salt fermented peppers with different bacteria concentrations;

FIG. 17 shows the change of reducing sugar of low-salt fermented peppers with different bacteria concentrations;

FIG. 18 shows amino acid nitrogen changes of low salt fermented peppers at different bacteria concentrations;

FIG. 19 shows the types and contents of volatile aroma substances of different fermented peppers;

FIG. 20 is a volatile aroma clustering heat map.

Detailed Description

The invention will be described in detail below with reference to the drawings in connection with embodiments. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.

The invention adopts SPSS26 to carry out single factor analysis of variance (LSD method and Duncan method) and significant difference comparison, p < 0.05 is significant difference, excel is used to carry out data processing and Origin 2022 mapping. Each experiment was repeated 3 times and the results are expressed as "x±s".

EXAMPLE 1 isolation of species

1.1 test materials

Capsicum annuum, salt, 1L fermentation jar, etc. are purchased from the source farmer market in the east of Hunan agricultural university.

1.2 test strains

Coli (Escherichia coli), staphylococcus aureus (staphylococcus aureus), salmonella (Salmonella) are supplied by the food science and technology institute microbiological laboratory at the university of hunan, agriculture.

1.3 test methods and results

1 preparation of Natural fermented Capsici fructus

Washing fresh red pod peppers with clear water for 2-3 times, airing in a ventilated and shady place until the surface is free of water, cutting into 1-2 cm small sections, adding 8% (w/w) edible salt, filling into a fermentation jar, sealing a jar cover with saline water, and standing at room temperature for 15-60 d.

2 isolation of species

Diluting 25g of naturally fermented pepper by ten times dilution method, collecting 100 μl of each gradient dilution, and uniformly coating on a solution containing 1% CaCO ₃ (W/V) MRS solid medium and PDA medium plates were incubated at 37℃and 28℃for 48-72 h, respectively. Observing colony morphology, picking out colony with obvious calcium dissolving ring on MRS solid culture medium, streaking and purifying for many times, streaking suspicious single colony on PDA culture medium on Bengalhon culture medium for 2-3 times until colony morphology is single, microscopic examination is free from impurity bacteria, transferring the separated strain after enrichment into 50% glycerol (1:1, V/V) tube, freezing at-80 ℃ and preserving at 4 ℃ test tube inclined plane for standby.

Preliminary identification of 13 species

Lactic acid bacteria: coating the strain after screening on Ca-containing strain ₂ CO ₃ Is cultured for 48 hours at a constant temperature of 37 ℃ and the size of a calcium-melting ring and the morphology of single colonies grown are observed. And (3) performing a hydrogen peroxide thixotropic enzyme test, picking single colonies, and performing gram staining microscopic examination to observe the staining condition of the thalli and recording.

Yeast: culturing on YPD medium for 48-72 hr, and observing colony color, texture, surface morphology, edge condition, etc. The bacterial cells were pelleted with basic methylene blue, and individual morphology of the yeast cells was observed under a microscope and asexual reproduction was recorded.

The results show that: from the fermented capsicum sample, 11 strains with calcium-dissolving ring, negative contact enzyme, no spore and gram positive are separated, and the strains are primarily judged as lactobacillus and respectively named as lactobacillus R ₁ ～R ₁₁ . The colony is opaque, raised or slightly raised, milky white, milky yellow or off-white, and when single colony is picked upA yogurt-like texture; the cell morphology is rod-shaped or short rod-shaped, single or paired, and is short-chain, and specific colony morphology and cell morphology are shown in fig. 1 and table 1.

TABLE 1 colony morphology and cell morphology of lactic acid bacteria strain 11

1.5.4 lactic acid bacterium Performance measurement

Preparation of bacterial suspension: single colonies on the slant medium stored in 1.5.2 were picked up to MRS broth medium, and were subjected to stationary culture at 37℃for 24 hours, followed by activation twice. Centrifuging the bacterial liquid at 4deg.C and 8000r/min for 10min, collecting bacterial cells, washing with 0.85% (W/V) sterile physiological saline for 2 times, adjusting bacterial liquid concentration by Maillard turbidimetry, and preserving at 4deg.C.

(1) Acid production energy measurement: take 10 ⁹ 200 mu L of CFU/mL bacterial suspension is injected into a 10mL centrifuge tube filled with 5mL MRS broth culture medium, and is cultured for 48 hours at 37 ℃, and the pH value is measured once at 0, 2, 4, 6, 8, 10, 12, 24, 36 and 48 hours respectively; meanwhile, the acidity of the fermentation broth is measured by referring to a phenolphthalein indicator method in national standard GB/T5009.239-2016, 1mL of culture broth after 24h fermentation is taken for centrifugation, distilled water is added for dilution to 30mL, 2-3 drops of 1g/L phenolphthalein are added as an indicator, 0.1mol/L NaOH standard solution is used for titration, the acidity of the lactobacillus fermentation broth is represented by Ji Ernie mol/L NaOH standard solution (DEG T), namely 1mL NaOH solution with the concentration of 0.1mol/L is consumed for each 100mL of fermentation broth, 1 DEG T is equivalent, a non-inoculated MRS broth culture medium is used as a blank control, 3 repetitions are added for each group, and the lactobacillus with fast and high acid production is screened.

The results show that: 11 strains of lactic acid bacteria R separated from fermented capsicum samples ₁ ～R ₁₁ Culturing for 48 hours, and measuring the pH value and titratable acidity, wherein the pH value change trend is shown in figure 2, and the final pH value and acidity value are shown in table 2; the pH value in the blank culture medium is always kept at about 5.39 within 48 hours, and R in 11 strains of lactic acid bacteria ₈ 、R ₁₀ 、R ₁₁ The final fermentation pH of the fermentation is above 4.0, the acidity is low, compared with other lactic acid bacteria, the acid production is poor, and the lactic acid bacteria R ₁ 、R ₂ 、R ₆ 、R ₇ 、R ₉ The pH value is still above 4.0 at 10h of fermentation, the acid production rate is general, and R ₃ 、R ₄ 、R ₅ The pH of the three strains of lactic acid bacteria is rapidly reduced to about 3.8 within the first 10 hours, the final pH is below 3.5 after 48 hours, the acidity is above 200 DEG T after 24 hours of fermentation, the acid production rate is high, the acid production is high, but after independent fermentation, the lactic acid bacteria R ₅ Acid production rate of R ₃ 、R ₄ Slow, so R is selected ₃ 、R ₄ These two strains of lactic acid bacteria were subjected to the next experiment.

TABLE 2 acid production capacities of different lactic acid bacteria

Note that: the data in the table are mean ± standard deviation of 3 replicates, all with one same lowercase letter after the same column of data, indicating that the difference is not significant (p >0.05, duncan's method).

(2) Antibacterial property measurement: escherichia coli, staphylococcus aureus and salmonella are taken as indicator bacteria, and the mixture is subjected to stationary culture for 24 hours at 37 ℃. Adopting an agar perforation bacteriostasis circle method to carry out bacteriostasis experiments on three lactobacillus R3, R4 and R5 with higher acid production selected by the experiment, and taking 10 ⁸ 100 mu L of CFU/mL indicator bacteria suspension is coated on NA culture medium, then a 6mm puncher is used for punching, 100 mu L of lactobacillus fermentation supernatant is added into the holes, MRS broth culture medium is used as a reference, 3 times of culture are repeated for each group, the culture is kept stand for 4 hours in a refrigerator at 4 ℃ before the liquid is fully diffused into agar, then the culture is carried out for 24 hours at 37 ℃, the diameter of a bacteriostasis ring is measured, and lactobacillus with pathogenic bacteria inhibiting function is screened.

As can be seen from Table 3 and FIG. 3, compared with the non-inoculated MRS broth, the fermentation supernatants of three lactic acid bacteria have obvious inhibitory effects on Escherichia coli, staphylococcus aureus and Salmonella, the difference is obvious (p < 0.05), and the lactic acid bacteria R ₃ The antibacterial agent has the best inhibition effect on escherichia coli, staphylococcus aureus and salmonella, and the antibacterial diameters are 19.02+/-0.87 mm, 19.77 +/-0.24 mm and 19.12+/-0.63 mm respectively. Lactic acid bacteria R ₄ 、R ₅ The bacteriostasis diameter is not greatly differentThe difference is not obvious (p is more than 0.05), but the antibacterial diameters are proved to be about 18 mm. To sum up, lactic acid bacteria R ₃ 、R ₄ 、R ₅ Has good inhibition effect on all three pathogenic bacteria.

TABLE 3 inhibition of pathogenic bacteria by different lactic acid bacteria fermentation broths

Note that: the data in the table are mean ± standard deviation of 3 replicates, where each row of data is followed by a single lower case letter, indicating that the difference is not significant (p >0.05, duncan's method).

1.5.5 Yeast Performance determination

The method is characterized in that 7 yeasts are separated from the fermented chilli and respectively named as yeasts J ₁ ～J ₇ . The colony forms are round bulges or slightly bulges, most of the colony forms have gloss or wetness, opaque white or milky white, dead cells are dark blue after microscopic examination after being dyed by methylene blue, living cells are colorless and transparent, vacuoles are visible, the forms are different, the cell forms are oval, round, elliptic or with false hyphae and the like, budding and division and reproduction are visible at one end or two ends, and the specific colony forms and cell forms are shown in fig. 4 and table 4.

Table 4 7 colony morphology and cell morphology description of Yeast strains

The performance of the saccharomycetes is detected as follows:

preparation of bacterial suspension: and (3) picking single colonies on the preserved slant culture medium to YPD liquid culture medium, performing stationary culture at 28 ℃ for 24 hours, and activating twice. Centrifuging the bacterial liquid at 4deg.C and 8000r/min for 10min, collecting bacterial cells, washing with 0.85% (W/V) sterile physiological saline for 2 times, regulating yeast amount by blood cell counting method, and preserving at 4deg.C.

(1) Gas production performance measurement: fermentation by Du's tubeIn this method, the Du's tube was filled with YPD liquid medium, and the tube was inverted into a test tube containing 10mL of YPD liquid medium, and sterilized at 121℃for 15 minutes. Take 10 ⁸ 200. Mu.L of CFU/mL bacterial suspension was injected into the above tubes, and the culture was allowed to stand at 28℃for 3 replicates per group. Every 12 hours, the amount of bubbles in the Du's tube accounting for the volume of the Du's tube is observed, and the saccharomycetes with serious gas production are eliminated.

As shown in Table 5, yeast J ₄ The gas production is the fastest, the gas production begins within 12 hours, the whole Du Shixiao pipe is filled after 48 hours, and then the saccharomycete J ₃ 、J ₇ After 36h of culture, gas is produced, the gas production reaches 1/2 and 1/4 respectively, and the residual saccharomycete J ₁ 、J ₂ 、J ₅ 、J ₆ No gas generation or a slow negligible gas generation during 48 hours of incubation.

TABLE 5 gas production in 48h for different yeasts

Note that: 0 is no gas generation, 1 is gas filling Du's tube, and the rest is gas accounting for Du's tube volume ratio.

(2) Measuring the yield and the property: culturing activated yeast at 28deg.C for 48 hr, observing, and recording whether the surface of the yeast is produced with uncut (a layer of white or off-white bacterial film formed at the interface of the culture medium liquid surface and air contact, with or without uncut and with or without precipitate), and eliminating the yeast with serious uncut.

As shown in FIG. 5 and Table 6, yeast J ₂ 、J ₃ 、J ₄ There are different degrees of yield of uncut, wherein yeast J ₄ The most serious of the yield is the yeast J ₃ Yeast J ₂ There is little uncut and uncut phenomenon, saccharomycete J ₁ 、J ₅ 、J ₆ 、J ₇ The uncut is not produced. Yeast J was found in tables 2 to 8 ₇ Gas production, thus adopt J ₁ 、J ₅ 、J ₆ The three yeasts were subjected to the next acid resistance test.

TABLE 6 different Yeast Pu yield conditions

Note that: + is generated by a small amount of uncut and++ is more produced, ++ is severe in the case of producing a uncut, and no uncut.

(3) Acid resistance measurement: preparing YPD liquid culture medium, and adjusting pH values of the YPD liquid culture medium to 3, 3.5, 4, 4.5 and 5 respectively by taking 1M/L lactic acid solution as a pH regulator. Mixing, packaging in test tube with 10mL, and sterilizing at 121deg.C for 15min. Take 10 ⁸ 200 mu L of CFU/mL bacterial suspension is injected into the test tubes, 3 replicates of each group are respectively treated by taking YPD liquid culture medium without bacteria as a blank control, and after the culture is carried out for 24 hours at the constant temperature of 28 ℃, a vortex oscillator oscillates for 30 seconds, and OD is measured ₆₀₀ And (5) obtaining and recording values, and screening out saccharomycetes with better acid resistance.

The results are shown in FIG. 6. Overall, OD decreases with decreasing pH ₆₀₀ The value also gradually decreased, but yeast J ₅ Compared with microzyme J ₁ Under acidic condition (pH is less than or equal to 4), the growth is slower, and OD ₆₀₀ The value drop rate is large, especially at pH value of 3, saccharomycete J ₅ OD of (d) ₆₀₀ The value is less than 0.5. Yeast J ₆ OD at different pH ₆₀₀ Values below 1.0 may be due to slower growth per se or less prone to growth under acidic conditions. While Yeast J ₁ OD at pH 3.5 ₆₀₀ OD at pH 1.56, pH 4 ₆₀₀ The values reach 1.98, which are all obviously higher than that of microzyme J ₅ 、J ₆ (p < 0.05). To sum up, consider yeast J ₁ Is used for the next step of fermentation pepper experiments.

Example 2

Fermenting the screened lactobacillus R3 and R4 and saccharomycete J1 under the condition of low salt (8% (W/W) salinity) by compounding single strain, measuring basic indexes such as pH, total acid, reducing sugar, amino acid nitrogen and the like by sensory evaluation, measuring the content of organic acid after fermentation by using high performance liquid chromatography, measuring volatile substances (Volatile Aroma Components and VOC) by using headspace solid-phase microextraction-gas chromatography-mass spectrometry, and carrying out differential analysis on the flavor quality of room-temperature natural fermentation pepper products (18% (W/W) high salinity), non-inoculation room-temperature natural fermentation peppers, non-inoculation fermentation peppers and inoculation peppers at 32 ℃ provided by X company by using cluster analysis and main component analysis, so as to eliminate the compound strain with poor fermentation quality.

1 test method

1.1 preparation of fermented Capsici fructus

Preparation of bacterial suspension: see example 1

And (3) raw material treatment: fresh, crisp, tender, fresh red, no rot and mildew, no mechanical damage and no plant diseases and insect pests are selected, the pod is washed by distilled water for 2 to 3 times after being removed, the pod is dried in the shade and ventilated place, cut into small sections of 1cm to 2cm, added with 8 percent (w/w) edible salt, and packaged into 100g of glass bottles which are boiled and sterilized at high temperature.

Control group: sterile room temperature natural fermentation Capsicum (CK) ₁ ) Fermented pepper (CK) without inoculation at 32 DEG C ₂ ) Room temperature natural fermentation Capsicum product (CK) provided by company X ₃ ) (18% (W/W) high salinity, natural fermenting at room temperature for 15d, and flavoring).

Treatment group: inoculating fermented capsicum group with R ₃ :J ₁ 、R ₄ :J ₁ 、R _3: R ₄ . The total inoculation amount is 6% (w/w), and the lactobacillus concentration is 1×10 ⁹ CFU/mL, yeast concentration of 1×10 ⁸ CFU/mL, strain ratio is 1:1, and fermentation is carried out at 32 ℃ in a sealing way.

The evaluation index was the same as in example 2, and the specific results were as follows:

1.2 sensory evaluation

After 15d fermentation, sensory evaluation was performed. With reference to and improvement of the standard NY/T1711-2020 "Green food Capsici preparation", a sensory panel was composed of 10 persons in the food professional background, and the scoring criteria are shown in the following table:

TABLE 7 sensory quality evaluation criteria for fermented Capsici fructus

As shown in Table 8, the sensory scores of the different fermented peppers were R ₃ :J ₁ ＞R ₄ :J ₁ ＞R ₃ :R ₄ ＞CK ₁ ＞CK ₂ 。R ₃ :J ₁ The overall score of the group is higher, 90.81 and 88.80 respectively, R ₃ :J ₁ The color of the group is good; and R is ₃ :J ₁ The sweet and sour flavor has typical lactic acid fermentation flavor, flower and fruit flavor and the like, is unique and complex in flavor and has moderate sour and salty taste. And R is R ₃ :J ₁ 、R ₄ :J ₁ In comparison, R ₃ :R ₄ Is 84.18 points. R is R ₃ :J ₁ 、R ₄ :J ₁ The comprehensive sensory score of the lactobacillus and microzyme fermenting and fermenting the same is higher than that of single strain fermenting, and is mainly reflected in the promotion of taste and aroma, so that the lactobacillus and microzyme co-fermenting sensory quality is better than that of single strain inoculating and fermenting, and the flower and fruit aroma generated by microzyme fermenting is added while the lactobacillus and microzyme fermenting has sour and spicy aroma.

TABLE 8 sensory scores of fermented peppers

Note that: different letters in the same column represent significant differences (p < 0.05), whereas no significant differences (p > 0.05) are present. And (3) injection: CK (CK) ₁ Natural fermentation of capsicum and CK at room temperature without inoculation ₂ Fermenting capsicum at 32deg.C without inoculating bacteria; r is R ₃ :J ₁ For inoculation of lactic acid bacteria R ₃ And Yeast J ₁ Fermented chilli, R ₄ :J ₁ For inoculation of lactic acid bacteria R ₄ And Yeast J ₁ Fermented chilli, R ₃ :R ₄ For inoculation of lactic acid bacteria R ₃ And lactic acid bacteria R ₄ And (5) fermenting the chillies.

1.3 basic index determination

The changes in the contents of pH, total acid, reducing sugar, amino acid nitrogen, etc. were measured every two days, and after 15d fermentation, the organic acid content was measured.

(1) And (3) pH measurement: the pH meter method is used for measuring with reference to GB/T5009.237-2016 (determination of the pH value of food safety national Standard food);

as shown in FIG. 7, CK was recovered with the increase of fermentation time ₁ 、CK ₂ The pH change after 15d fermentation was not significant (p > 0).05). The pH value of other groups starts to rapidly decrease in 3d of fermentation until the pH value after 12d of fermentation is stable, R ₃ :J ₁ The pH value of (C) is reduced to 3.77, R ₄ :J ₁ The pH value of (C) is reduced to 3.85, R ₃ :R ₄ The pH of (c) drops to 3.79. From this, it was found that the lactic acid bacteria R were under the same conditions as the total inoculum size ₄ The fermentation acid production speed is lower than that of lactic acid bacteria R ₃ 。

(2) Total acid determination: the determination is carried out by adopting an acid-base indicator titration method in GB/T12456-2021 determination of total acid in food safety national standard food;

From FIG. 8, the change trend of the total acid of different fermented peppers, R, can be seen ₃ :J ₁ 、R ₄ :J ₁ 、R ₃ :R ₄ Significantly reduced (p < 0.05), R ₃ :J ₁ 、R ₄ :J ₁ 、R ₃ :R ₄ The total acid content of the catalyst reaches 0.74g/100g, 0.70g/100g and 0.73g/100g respectively. R after 15d fermentation with the total inoculum size unchanged, the homolactic fermentation ₃ :R ₄ The acid production rate is not higher than that of the lactobacillus and saccharomycete co-fermentation group R ₃ :J ₁ High (p > 0.05). In addition, homolactic group R ₃ :R ₄ There was no significant difference (p > 0.05) in total acid compared to the fermentation of the lactic acid bacteria (with the total inoculum size unchanged), probably due to competition and inhibition of lactic acid production by the lactic acid bacteria.

(3) Reducing sugar determination: the determination is carried out by adopting a direct titration method in GB/T5009.7-2016 (determination of reducing sugar in food safety national standard food);

as can be seen from FIG. 9, the reducing sugar content in the capsicum decreases with fermentation, R ₃ :J ₁ 、R ₄ :J ₁ 、R ₃ :R ₄ The content change trend of the reducing sugar is consistent, the reducing sugar is consumed by lactobacillus and saccharomycetes in the initial fermentation period, R ₃ :J ₁ Reducing sugar content to 1.72g/100g, R ₄ :J ₁ Reducing sugar content to 1.80g/100g, R ₃ :R ₄ The reducing sugar content was reduced to 1.87g/100g, indicating R ₃ :J ₁ Degree of utilization of reducing sugarFaster, faster fermentation. After 12d fermentation, microorganisms in the capsicum are gradually inhibited due to accumulation of acid, and the reducing sugar content is stable. And CK (CK) ₂ As the bacteria are not inoculated, the reducing sugar still continuously descends after 12d of fermentation, and CK ₂ The decrease is faster, and the temperature of 32 ℃ is probably more favorable for the growth of microorganisms than the natural fermentation condition at room temperature.

(4) Amino acid nitrogen determination: the determination is carried out by adopting an acidometer-formaldehyde titration method in GB/T5009.235-2016 determination of amino acid nitrogen in food safety national standard food;

as shown in FIG. 10, 3d before fermentation, the growth metabolism of microorganisms breaks down proteins due to their increased number of microorganisms, resulting in an increase in the nitrogen content of amino acid states, where R ₃ :J ₁ The maximum rise is 0.323g/100g, R ₄ :J ₁ 0.313g/100g, R ₃ :R ₄ 0.307g/100g, R ₃ :R ₄ Is lower than the other inoculated fermentation groups, indicating that this group utilizes protein more slowly than the other groups. In addition, the amino acid nitrogen generated by double-strain fermentation is higher than the amino acid nitrogen generated by single lactobacillus fermentation, which indicates that the double-strain fermentation can improve the quality of the fermented peppers. The amino acid nitrogen is not increased substantially in the middle and later stages of fermentation, and may be caused by the fact that the microorganism consumes a part of the amino acid nitrogen as a nitrogen source in the middle and later stages of fermentation.

(5) Determination of organic acids: reference is made to GB 5009.157-2016 "determination of organic acids in food" and slight modifications.

Sample treatment: the fermented chilli sample is beaten into homogenate, 1.50g is weighed and placed in a 50mL volumetric flask for constant volume. Then extracting in water bath at 70deg.C for 20min, and cooling to room temperature. And filtering to remove capsicum residue, centrifuging the filtrate at 4000r/min for 15min, collecting supernatant, filtering with 0.22 μm water-based filter membrane, and injecting into a liquid-phase sample bottle for analysis by high performance liquid chromatograph.

Standard curve preparation: standard solutions of oxalic acid with mass concentration of 500 mug/mL, tartaric acid of 12500 mug/mL, malic acid of 25000 mug/mL, lactic acid of 25000 mug/mL, acetic acid of 25000 mug/mL, citric acid of 25000 mug/mL and succinic acid of 62500 mug/mL are respectively prepared, diluted by 25 times, 50 times, 125 times, 250 times and 500 times respectively by 0.1% phosphoric acid solution to obtain organic acid standard solutions with different concentrations, and the organic acid standard solutions are filtered by a 0.22 mu m water-based filter membrane and then injected into a liquid phase sample bottle for analysis by a high performance liquid chromatograph.

Chromatographic conditions: mobile phase 0.1% phosphoric acid solution: methanol=97.5:2.5 (V/V); the flow rate is 1.0mL/min; column temperature is 40 ℃; the sample injection amount is 20 mu L; the detection wavelength was 210nm.

Qualitative and quantitative: and (3) taking the retention time of the organic acid standard solution as a qualitative standard, and carrying out identification analysis on the organic acid in the fermented chilli. And drawing a standard curve by taking the concentration of the organic acid standard solution as an abscissa and the area of a chromatographic peak as an ordinate, and calculating the content of the organic acid in the fermented chilli by using a standard curve equation.

As can be seen from Table 9, the double seed inoculated fermentation group R ₃ :J ₁ 、R ₄ :J ₁ 、R ₃ :R ₄ The organic acid content of (2) is obviously higher than that of the natural fermentation group CK ₁ 、CK ₂ 、CK ₃ . Compared with single strain fermentation, R ₃ :J ₁ 、R ₄ :J ₁ 、R ₃ :R ₄ Is significantly increased (p<0.05 Where R is ₃ :J ₁ The highest lactic acid content in the product is 84.74g/kg, R ₃ :R ₄ The citric acid content in the product is high and reaches 10.85g/kg. R is R ₃ :J ₁ The total amount of the organic acid is up to 158.50g/kg, and then R is ₃ :R ₄ 85.05g/kg, possibly R ₃ And R is ₄ The lactobacillus has competitive inhibition effect. R is R ₄ :J ₁ Is lower than R ₃ :J ₁ From this, it can be seen that lactic acid bacteria R ₄ The acid-producing capacity of fermentation is lower than that of lactobacillus R ₃ 。

TABLE 9 organic acid content in fermented Capsici fructus

Note that: "-" indicates undetected; the different letters in the same row represent significant differences (p < 0.05), whereas no significant differences are present(p > 0.05). And (3) injection: CK (CK) ₁ Natural fermentation of capsicum and CK at room temperature without inoculation ₂ Fermenting Capsici fructus and CK at 32deg.C without inoculating bacteria ₃ Fermented pepper products offered for company X; r is R ₃ :J ₁ For inoculation of lactic acid bacteria R ₃ And Yeast J ₁ Fermented chilli, R ₄ :J ₁ For inoculation of lactic acid bacteria R ₄ And Yeast J ₁ Fermented chilli, R ₃ :R ₄ For inoculation of lactic acid bacteria R ₃ And lactic acid bacteria R ₄ And (5) fermenting the chillies.

1.4 volatile aroma determination

Solid phase microextraction conditions: 3.0g of fermented chilli sample is weighed in each group and placed in a 20mL headspace bottle, 3mL of saturated NaCl solution and 10 mu L of internal standard 2-octanol (0.16 mg/mL) are added, the mixture is balanced in a constant temperature water bath kettle at 80 ℃ for 15min, water bath is continued, an extraction head is pushed to be about 1cm away from the liquid level, the mixture is quickly pulled out after constant temperature absorption for 40min, and the mixture is placed in a sample inlet and analyzed at 250 ℃ for 5min.

Gas chromatography conditions: the chromatographic column is DB-5MS capillary column (30 m×0.25mm×0.25 μm); heating program: heating the initial temperature to 220 ℃ at 5 ℃/min (keeping for 3 min), and heating to 250 ℃ at 10 ℃/min (keeping for 3 min); the flow rate of the high-purity nitrogen is 1mL/min.

Mass spectrometry conditions: an EI ion source, electron energy and temperature of 70eV and 230 ℃; the interface temperature is 250 ℃; the mass scanning range is 35-400 m/z, and sample injection is not split.

As can be seen from FIG. 11, CK ₁ 71 volatile aroma substances, CK, were detected ₂ 78 kinds of CK were detected ₃ 60 kinds of R are detected ₃ :J ₁ 92 kinds of R are detected ₄ :J ₁ 80 kinds of R are detected ₃ :R ₄ 74 kinds of R are detected ₃ :J ₁ The most volatile aroma species. The results demonstrate that inoculation of pure lactic acid bacteria to ferment capsicum reduces the variety of volatile aroma substances and that the volatile aroma substances increase after co-fermentation of lactic acid bacteria and yeast.

As can be seen from the cluster analysis of FIG. 12, CK ₃ The ratio of the esters is 33.46%, CK ₁ 、CK ₂ The esters are lower (the ratio is 16.29 to 18.10 percent), and the esters after inoculation and fermentation are obviously raised(p＜0.05)，R ₃ :J ₁ 、R ₄ :J ₁ 、R ₃ :R ₄ The ester accounts for 40.77%, 32.26%, 23.75% and R ₃ :J ₁ The highest ester content. Wherein ethyl acetate, myristyl acetate, ethyl decanoate, ethyl elaidic acid, isoamyl decanoate, cis-3-hexenyl butyl ester, cis-7-tetradecyl acetate and R ₃ :J ₁ Positive correlation is established. Hexadecyl acetate, ethyl myristate, ethyl pentadecanoate, isoamyl decanoate and the like and R ₄ :J ₁ Positive correlation is established. At R alone ₃ :R ₄ Stearyl acetate and ethyl linolenate were detected.

Acid in CK ₃ The highest proportion of the total organic acid is 51.62%, and the content of acids such as benzoic acid, sorbic acid and the like is mainly used as CK ₃ The flavor is blended again after natural fermentation, so that the defect of sour taste is overcome, and the flavor of the prepared fermented chilli product is more perfect. Compared with single strain fermentation, the acid in the compound fermentation of different strains is reduced, R in the single strain fermentation ₃ 、R ₄ The content of the acid substances in the fermentation broth is 32.37 percent and 29.17 percent respectively, and the ratio of the acid substances after the compound fermentation of different strains is 18.12 percent to 29.67 percent, which is probably due to the mixed inoculation of saccharomycetes, so that the acid substances and alcohols are subjected to esterification reaction, part of acid is consumed, and the content of esters is increased. Wherein pelargonic acid, octanoic acid and R ₃ :J ₁ Has positive correlation and fruit fragrance. Lauric acid, undecylenic acid, and the like, and R ₄ :J ₁ In positive correlation, palmitic acid, pentadecanoic acid, 8-methylnon-6-enoic acid is in R ₃ :R ₄ The content of the components is higher.

Alcohols at R ₃ :J ₁ The ratio of the R to the R is 12.75 percent ₄ :J ₁ The ratio of the R to the R is 13.01 percent ₃ :R ₄ The ratio of the medium to the CK is 8.74 percent ₁ (9.65％)、CK ₂ (12.76％)、CK ₃ Compared with (2.85%), the inoculation of lactobacillus and saccharomycetes can effectively increase the content of alcohol substances, CK ₂ The higher alcohols of (2) are probably the result of fermentation at 32℃for other microbial production. Benzyl alcohol, linalool, n-heptanol, trans-nerolidol, 2-hexyl-1-decanol, 3-methyl-2-octanol, and the likeAt R ₃ :J ₁ And R is ₄ :J ₁ More prominent. R is R ₃ :R ₄ There are also small amounts of oleyl alcohol, cyclododecyl methanol, etc.

R ₃ :J ₁ The content of n-hexanal, nonanal, phenylacetaldehyde, tridecyl aldehyde, heptadecyl aldehyde, (E) -2-hexenal, (Z) -2-heptenal and other substances is higher. R is R ₄ :J ₁ The proportion of the medium olefins is more than 2%, and the longifolene, (-) -isocaryophyllene and the beta-sesquiphellandrene are positively correlated. R is R ₃ :R ₄ The ketone is more, the ratio is 7.74%, such as geranylacetone, beta-ionone, etc.

1.5 calculation of qualitative and quantitative and relative odor Activity values of volatile aroma components

Qualitative: the mass spectrum data of the aroma substances are compared by NIST library 14, NIST library 17 and essence and spice standard spectrum library, the matching degree is more than or equal to 85 percent and is used as a qualitative substance identification standard, and literature data is consulted to check and confirm each volatile aroma substance in the fermented peppers. Quantification: by internal standard method ^[64,65] By adding 10 mu L of 2-octanol (0.16 mg/mL) as an internal standard substance to the fermented pepper sample in the above test, the peak area, mass concentration and volume of the internal standard substance were known, and then the relative content thereof was calculated from the mass spectrum peak area ratio of each component to the internal standard substance, specifically calculated as shown in the following formula (1). Relative odor activity value calculation: the fragrance activity value (Relative Odor Activity Values, rOAVs) is calculated from the mass concentration of each volatile fragrance component, as calculated by the following formula (2):

And (3) injection: c-the relative content of the volatile components (μg/Kg);

A ₁ -peak areas of the components;

A ₂ internal standard peak surfaceAccumulating;

C ₁ -internal standard mass concentration (μg/mL);

V ₁ -internal standard volume (mL);

m-sample mass (Kg);

t-the fragrance threshold concentration of the volatile component in water (μg/Kg).

The quality of the flavor of the fermented peppers cannot be judged from the types and the contents of the aroma substances, and the relative odor activity value rOAVs, which is an important index for evaluating the contribution of each aroma component to the flavor of the fermented peppers, needs to be calculated for comprehensive analysis. The higher the rOAVs value, the greater the contribution, and the fragrance components with the rOAVs value smaller than or equal to 0.1 are generally judged to be the action of coordinating the fragrance, and the rOAVs value larger than or equal to 1 is called as key fragrance components and has obvious influence on the flavor. Calculating rOAVs according to formula (2), wherein the result is shown in Table 10, and fermenting the capsicum differently to obtain 42 volatile aroma substances (rOAVs not less than 0.1) in total ₁ There are 24 kinds of CK ₂ There are 25 kinds of CK ₃ There are 15 kinds of R ₃ :J ₁ There are 36 kinds of R ₄ :J ₁ There are 25 kinds of R ₃ :R ₄ There are 20, inoculating fermentation group R ₃ :J ₁ The key aroma substances of rOAVs > 1 are the most.

TABLE 10 rOAVs values of different fermented Capsici fructus volatile substances ^[84,87]

And (3) injection: "-" indicates undetected; CK (CK) ₁ Natural fermentation of capsicum and CK at room temperature without inoculation ₂ Fermenting Capsici fructus and CK at 32deg.C without inoculating bacteria ₃ Fermented pepper products offered for company X; r is R ₃ :J ₁ For inoculation of lactic acid bacteria R ₃ And Yeast J ₁ Fermented chilli, R ₄ :J ₁ For inoculation of lactic acid bacteria R ₄ And fermentingMother strain J ₁ Fermented chilli, R ₃ :R ₄ For inoculation of lactic acid bacteria R ₃ And lactic acid bacteria R ₄ And (5) fermenting the chillies.

7.3 principal component analysis of volatile aroma substances

The aroma components with rOAVs greater than 1 were analyzed by PCA using Origin and plotted, and as shown in FIG. 12, the variance contribution ratios of the main component PC1 and the main component PC2 were 47.0% and 28.7%, respectively, and the cumulative variance contribution was 75.7%, which can reflect the main characteristics between fermented peppers. CK (CK) ₁ And CK (CK) ₂ The negative half shafts distributed on PC1 and PC2, and the 1-octanol and the geraniol have higher loads on the negative half shafts, which indicates that the content in the natural fermentation group is higher. CK (CK) ₃ Distributed on the negative half shaft of PC1 and the positive half shaft of PC2, and the mixture of lauric acid ethyl ester, methyl salicylate, caryophyllene and alpha-terpineol is used in CK ₃ Nearby distribution, demonstrating that these species are present in CK ₃ The content of the components is higher. R is R ₃ :J ₁ The substances distributed nearby are more, hexyl acetate, linalool, phenethyl alcohol, undecanal, n-hexanal and D-limonene have higher load on the positive half shafts of PC1 and PC2, R ₄ :J ₁ Distributed on the positive half axis of PC1 and the negative half axis of PC2, phenylacetaldehyde, trans-2-octenal and the like are distributed on R ₄ :J ₁ The content of the components is higher.

In summary, the results are as follows:

1. sensory evaluation found that R ₃ :J ₁ The comprehensive sensory score of (2) is 88.80, and compared with the single strain fermentation sensory score, the taste and the aroma are improved, and the sensory quality of the lactobacillus and the microzyme co-fermentation is better.

2. And measuring the pH value change of the low-salt fermented peppers compounded by different strains. And CK (CK) ₁ 、CK ₂ In comparison, R ₃ :J ₁ The pH value of (C) is reduced to 3.77, the ratio R ₄ :J ₁ And R is ₃ :R ₄ Is low.

3. And measuring the total acid change of the compound low-salt fermented peppers of different strains. R is R ₃ :J ₁ The total acid content of the catalyst reaches 0.74g/100g, and the acid production effect is best.

4. And measuring the reducing sugar change of the compound low-salt fermented peppers of different strains. R is R ₃ :J ₁ Reducing sugar content to 1.72g/100g, R ₃ :J ₁ The utilization degree of reducing sugar is faster, and the fermentation is faster.

5. And (3) measuring the amino acid nitrogen change of the compound low-salt fermented peppers of different strains. R is R ₃ :J ₁ The maximum increase in nitrogen content in the amino acid form of (2) is 0.323g/100g, R ₃ :R ₄ Amino acid nitrogen is lower than other inoculated fermentation groups.

6. And (5) measuring the organic acid content of the low-salt fermented peppers compounded by different strains. R is R ₃ :J ₁ The organic acid content of (2) reaches 158.50g/kg. Compared with single strain fermentation, the lactic acid content is significantly increased (p <0.05)。

7. And (5) measuring volatile aroma substances of the low-salt fermented peppers compounded by different strains. R is R ₃ :J ₁ The number of volatile aroma types is 92 at the maximum. R is R ₃ :J ₁ The highest ester content was 40.77%. There are 36 key aroma substances (rOAVs > 1).

8. By principal component analysis, R ₃ :J ₁ Most volatile aroma substances in (a) linalool (rovs=40.96), n-hexanal (rovs=12.99), hexyl acetate (rovs=9.87), undecanal (rovs=3.90), phenethyl alcohol (rovs=2.76), D-limonene (rovs=2.01) as R ₃ :J ₁ Key aroma substances of (2). To sum up, lactic acid bacteria R ₃ And Yeast J ₁ The quality of the co-fermented aroma is optimal.

Example 3

Materials and methods are the same as in example 2.

The test method is as follows:

1 fermentation seed identification

1 fermentation seed identification

The lactobacillus R with the best fermentation quality is screened ₃ And Yeast J ₁ After molecular biological identification and strain culture for 12 hours, the genomic DNA of the strain is extracted by using an Ezup column type bacteria/fungus genomic DNA extraction kit, and the lactobacillus is prepared by using a 16S rDNA bacterial universal primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3',1492R:5'-GGTTACCTTGTTACGACTT-3' PCR amplification; the yeast adopts a fungus universal primer NL1:5'-GCATATCAATAAGCGGAGGAAAAG-3', NL4:5'-GGTCCGTGTTTCAAGACGG-3' PCR amplification is carried out on the 26Sr DNA D1/D2 region gene, the PCR reaction system and the PCR reaction conditions are shown in tables 18 and 19 below, the PCR product is taken after the amplification and subjected to 1% agarose gel electrophoresis purification, and the PCR product is subjected to sequence alignment by the division of biological engineering (Shanghai) to identify the species. And (3) carrying out homology analysis and comparison on the sequencing result with the corresponding sequence of a known strain in a GenBank nucleic acid sequence database by using Blast software, and establishing a phylogenetic tree by using MEGA 5.0 software.

TABLE 11PCR reaction System

TABLE 12PCR reaction conditions

As shown in FIGS. 13 and 14, strain R ₃ And J ₁ After purification by 1% agarose gel electrophoresis, R ₃ 、J ₁ A single band with distinct specificity appears at 1500bp and 500bp, respectively. Sequencing and analyzing by biological engineering (Shanghai) Co., ltd to obtain 16S rDNA gene sequence and 26S rDNA gene sequence, and adding R ₃ 、J ₁ The strain gene sequence was uploaded to BLAST, and similarity analysis was performed by searching for a similar strain sequence having high homology using genbank, and a phylogenetic tree was constructed using Neighbor-Joining (N-J) method in MEGA X software. The results show that R ₃ The homology of the strain and lactobacillus plantarum is 99%, and R is identified ₃ The strain was lactobacillus plantarum Lactiplantibacillus plantarum (GenBank accession No. OQ 847791). J (J) ₁ The homology of the strain and the strain of the strain Saccharomyces cerevisiae Meng Maiye is 100%, and J is identified ₁ The strain was Saccharomyces cerevisiae Meyerozyma guilliermondii, also Meng Maiye (GenBank accession number OQ 851829).

Lactobacillus plantarum Lactobacillus plantarum R is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27361, the preservation time of 2023, 5 and 17 days, and the preservation address of North Chen Xiyu No. 1 and 3 in the Chaoyang area of Beijing city.

The strain Meng Maiye microzyme Meyerozyma guilliermondii J1 is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.27360, the preservation time is 2023 years, 5 months and 17 days, and the preservation address is North Chen Xiyu No. 1 and 3 in the Chaoyang region of Beijing city.

2 preparation of fermented Capsici fructus

Preparation of bacterial suspension: see example 2

And (3) raw material treatment: fresh, crisp, tender, fresh red, no rot and mildew, no mechanical damage and no plant diseases and insect pests are selected, the pod is washed by distilled water for 2 to 3 times after being removed, the pod is dried in the shade and ventilated place, cut into small sections of 1cm to 2cm, added with 8 percent (w/w) edible salt, and packaged into 100g of glass bottles which are boiled and sterilized at high temperature.

Control group: sterile room temperature natural fermentation Capsicum (CK) ₁ ) Fermented pepper (CK) without inoculation at 32 DEG C ₂ ) Room temperature natural fermentation Capsicum product (CK) provided by company X ₃ ) (18% (W/W) high salinity, natural fermenting at room temperature for 15d, and flavoring).

Treatment group: lactic acid bacteria R ₃ And Yeast J ₁ Adjusting to different bacteria concentrations, and respectively adjusting lactobacillus concentration (yeast concentration) to 1×10 ⁷ CFU/mL(1×10 ⁶ CFU/mL)、1×10 ⁸ CFU/mL(1×10 ⁷ CFU/mL)、1×10 ⁹ CFU/mL(1×10 ⁸ CFU/mL)、1×10 ¹⁰ CFU/mL(1×10 ⁹ CFU/mL) (hereinafter 10% ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ Represents fermentation groups with different bacteria concentrations), the total inoculum size is 6% (W/W), the strain ratio is 1:1, and the fermentation is carried out at the temperature of 32 ℃ in a sealing way.

The performance evaluation was the same as in example 2, and the results were as follows:

2.1 sensory evaluation of Low salt fermented Capsici fructus with different bacteria concentrations

The sensory evaluation results are shown in Table 13. In color and luster, 10 ⁹ :10 ⁸ ＞10 ⁸ :10 ⁷ ＞10 ¹⁰ :10 ⁹ ＞10 ⁷ :10 ⁶ ＞CK ₁ ＞CK ₂ In flavor, 10 ⁹ :10 ⁸ ＞10 ¹⁰ :10 ⁹ ＞10 ⁸ :10 ⁷ ＞10 ⁷ :10 ⁶ ＞CK ₂ ＞CK ₁ On aroma, 10 ⁹ :10 ⁸ ＞10 ¹⁰ :10 ⁹ ＞10 ⁸ :10 ⁷ ＞10 ⁷ :10 ⁶ ＞CK ₁ ＞CK ₂ In terms of brittleness, CK ₁ ＞10 ⁷ :10 ⁶ ＞10 ⁸ :10 ⁷ ＞10 ⁹ :10 ⁸ ＞10 ¹⁰ :10 ⁹ ＞CK ₂ Overall, sensory score of 10 ⁹ :10 ⁸ ＞10 ⁸ :10 ⁷ ＞10 ¹⁰ :10 ⁹ ＞10 ⁷ :10 ⁶ ＞CK ₁ ＞CK ₂ 。10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ The group has the phenomenon of incomplete fermentation and maturation, has lighter fermentation smell and better brittleness. 10 ¹⁰ :10 ⁹ Groups were below 10 on sensory scores ⁹ :10 ⁸ Group of 10 ¹⁰ :10 ⁹ The sourness is too intense after group fermentation, the brittleness is obviously reduced, and the sensory quality is reduced. 10 ⁹ :10 ⁸ The group sensory score was the best, 89.26 points. From this, it is found that the flavor and taste of the inoculated and fermented pepper are improved, but the crispness is also reduced.

TABLE 13 sensory scores of fermented peppers

Note that: different letters in the same column represent significant differences (p < 0.05), whereas no significant differences (p > 0.05) are present. CK (CK) ₁ Natural fermentation of capsicum and CK at room temperature without inoculation ₂ Fermenting capsicum at 32deg.C without inoculating bacteria; 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ Is R ₃ :J ₁ Fermented peppers with different bacteria concentrations.

2.2 pH variation of Low salt fermented Capsici fructus with different bacteria concentrations

As shown in FIG. 15, the pH of the capsicum decreases as the fermentation proceeds, and the capsicum CK is fermented without inoculation ₁ 、CK ₂ The pH value of (C) is slowly decreased, and finally, the pH value is about 4.36. Next, 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ The pH value of the fermented peppers in the group is reduced to 4.19 and 4.14, and the pH value is not reduced after 9d fermentation. Compared with the first three groups, 10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ The pH value of the fermented peppers in the group is obviously reduced (p is less than 0.05) to 3.75 and 3.56 respectively, and the pH value of the fermented peppers after 12d fermentation is stable.

2.3 Total acid changes in Low salt fermented Capsici fructus at different bacteria concentrations

As shown in FIG. 16, the pepper CK is fermented without bacteria ₁ 、CK ₂ The final total acid content of (2) is about 0.34g/100g, and the total acid content is slowly increased. As a result, the fermentation acid production rate was faster and the pH was lower as the concentration of bacteria increased. 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ The total acid of the fermented peppers in the group is raised to 0.39g/100g, 0.48g/100g,10 ⁷ :10 ⁶ The total acid was slightly reduced after 9d fermentation, possibly with insufficient lactic acid bacteria, resulting in reduced acid production in the later stages. 10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ The total acid content of the fermented peppers in the group reaches 0.74g/100g and 0.82g/100g respectively, the pH value is slightly reduced after 12d fermentation, and the excessive amount of lactic acid bacteria and the accumulation of lactic acid content in the later period possibly lead to the competitive inhibition and acid inhibition of strain growth metabolism, so that the acid yield is reduced and the consumption of acid substances is reduced.

2.4 Low salt fermented Capsici fructus reducing sugar changes with different bacteria concentrations

As shown in FIG. 17, the capsicum CK is naturally fermented at room temperature ₁ The reducing sugar of (2) is reduced to 1.92g/100g, and the trend is close to 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ The group fermentation of the capsicum is greatly influenced by external environmental factors by natural fermentation at room temperature, and the fermentation quality is uncontrollable. 10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ The fermented capsicum reducing sugar of the group is respectively reduced to 1.81g/100g and 1.54g/100g. Fermentation 1After 5d, fermenting the capsicum CK at 32 ℃ without inoculating bacteria ₂ Is 1.58g/100g and 10 g ¹⁰ :10 ⁹ The fermented peppers of the group had no significant difference (p > 0.05). In summary, as the concentration of bacteria increases, the consumption rate of reducing sugar increases. The utilization rate of reducing sugar is low, so that the capsicum is easy to ferment immature, and the green taste is obvious; since reducing sugar is consumed too much and capsicum becomes soft and peracid or the like is likely to occur, it is extremely important to select a proper bacteria concentration for fermentation.

2.5 amino acid Nitrogen changes in Low salt fermented Capsici fructus at different bacteria concentrations

As shown in fig. 18, as the concentration of bacteria increases, the protein utilization rate of the microorganism in the capsicum increases, and the final amino acid nitrogen content is from large to small: CK (CK) ₂ ＞10 ¹⁰ :10 ⁹ ＞10 ⁹ :10 ⁸ ＞CK ₁ ＞10 ⁸ :10 ⁷ ≥10 ⁷ :10 ⁶ 。10 ⁷ :10 ⁶ The amino acid nitrogen content of the group was the lowest, 0.287g/100g. Next, 10 ⁸ :10 ⁷ Group, 0.291g/100g, description 10 ⁷ :10 ⁶ Group sum 10 ⁸ :10 ⁹ The group of fermented peppers has fewer microorganisms and weaker proteolytic action.

2.6 analysis of organic acids of Low-salt fermented Capsici fructus with different bacteria concentrations

As can be seen from Table 14, the organic acid content of the fermented capsicum is 10 from high to low ⁹ :10 ⁸ ＞10 ¹⁰ :10 ⁹ ＞10 ⁸ :10 ⁷ ＞CK ₃ ＞10 ⁷ :10 ⁶ ＞CK ₂ ＞CK ₁ ，10 ⁷ :10 ⁶ And 10 ⁸ :10 ⁷ The organic acid content of the group is significantly lower than 10 ⁹ :10 ⁸ And 10 ¹⁰ :10 ⁹ Group (p < 0.05) in which oxalic acid was not detected, which may be 10 ⁷ :10 ⁶ And 10 ⁸ :10 ⁷ The amount of lactic acid bacteria in the fermentation is insufficient, and the minimum concentration of bacteria required for fermentation is not started, thereby resulting in insufficient yield of sour substances. 10 ¹⁰ :10 ⁹ The organic acid content ratio of 10 ⁹ :10 ⁸ Low group, indicating that when the amount of lactobacillus reaches a certain amount, the yield of organic acidDescending.

TABLE 14 organic acid content in fermented Capsici fructus

Note that: "-" indicates undetected; different letters in the same row represent significant differences (p < 0.05), whereas no significant differences (p > 0.05) are present. CK (CK) ₁ Natural fermentation of capsicum and CK at room temperature without inoculation ₂ Fermenting Capsici fructus and CK at 32deg.C without inoculating bacteria ₃ Fermented pepper products offered for company X; 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ Is R ₃ :J ₁ Fermented peppers with different bacteria concentrations

2.7 analysis of volatile aroma substances of low-salt fermented peppers with different bacteria concentrations

2.7.1 Cluster analysis of volatile aroma substances

As can be seen from fig. 19, 10 ⁷ :10 ⁶ Is composed of 83 volatile fragrant substances (10) ⁸ :10 ⁷ Is composed of 74 kinds of 10 ⁹ :10 ⁸ 92 kinds, 10 ¹⁰ :10 ⁹ There are 82 kinds of groups. 10 ⁷ :10 ⁶ The group contains higher alkane and ester content which respectively reach 29.64 percent and 21.56 percent. 10 ⁸ :10 ⁷ The group is relatively high in esters and alkanes, 27.53% and 26.67% respectively. 10 ⁹ :10 ⁸ The group has the highest ratio of esters to acids of 40.77% and 19.84%.10 ¹⁰ :10 ⁹ The content of esters and acids in the group is higher and reaches 27.42 percent and 24.04 percent respectively. From this, it is found that the co-fermentation of lactic acid bacteria and yeast mainly includes esters, and acids and esters gradually increase as the concentration of bacteria increases.

As can be seen from FIG. 20, the differences of the volatile aroma components of the fermented peppers with different bacteria concentrations were studied by cluster analysis, as shown in FIGS. 5-9, for red of different fermented pepper groupsThe blue correlation boundary is obvious, wherein the volatile aroma substances are positively correlated and are combined with CK ₃ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ . Wherein CK is ₃ Is positively correlated with ethyl elaidic acid, ethyl linoleate, ethyl palmitate, ethyl 9-hexadecenoate, geraniol, alpha-terpineol, sorbic acid, benzoic acid, caryophyllene, dihydro-beta-ionol, and the like. 10 ⁷ :10 ⁶ Is positively correlated with substances such as hexyl isovalerate, hexyl isobutyrate, 12-methyl-tridecanol and the like. 10 ⁸ :10 ⁷ Is positively correlated with beta-bisabolene, 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H) -pyran-4-one, (+) -dihydro-carvone, heptadecane, etc. 10 ⁹ :10 ⁸ Is positively correlated with substances such as isoamyl decanoate, hexyl acetate, myristyl acetate, 2-hexyl-1-decanol, nonanal, n-hexanal, D-limonene and the like. 10 ¹⁰ :10 ⁹ Is positively correlated with leaf alcohol, palmitol, n-hexanol, undecylenic acid, 4-methyl valeric acid, 4-methyl-1-pentanol, ethyl myristate, ethyl salicylate, etc. Wherein linalool, phenethyl alcohol, trans-nerolidol, geranylacetone, cis-3-hexenyl butyl ester, cis-7-tetradecyl acetate and the like and 10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ There is a high correlation, probably Lactobacillus plantarum R ₃ And Klebsiella xylosoensis J Meng Maiye ₁ The characteristic aroma substances of the co-fermentation endow the fermented peppers with unique fresh fruit aroma, green grass aroma, rose aroma, magnolia aroma and other odors.

Principal component analysis of volatile aroma substances

The results show that CK1 and CK2 are distributed on the negative half shafts of PC1 and PC2, and the effect of temperature on volatile aroma substances is small because the difference between the room-temperature naturally fermented peppers (CK 1) and the 32-DEG C naturally fermented peppers (CK 2) is not large. The result of the principal component analysis of CK3 is the same as before. 10 ⁷ :10 ⁶ And 10 ¹⁰ :10 ⁹ Near the origin point, the score is lower, indicating a comparison of 10 ⁸ :10 ⁷ Group sum 10 ⁹ :10 ⁸ Groups of less characteristic aroma substances。10 ⁸ Around the group are distributed hexyl isovalerate, guaiacol, trans-2-octenal, etc. 10 ⁹ :10 ⁸ The group mainly focuses on the normal half shafts of PC1 and PC2, and aroma substances such as nonanal, hexyl acetate, linalool, n-hexanal, 4-ethylphenol, undecanal, D-limonene and the like are distributed around the groups, which is shown in 10 ⁹ :10 ⁸ The content of the medium is higher, which is similar to the principal component analysis of the previous R3:J1 group.

To sum up, 1, for lactic acid bacteria R ₃ And Yeast J ₁ 16S rDNA and 26S rDNA were sequenced, respectively. Identification of R ₃ The strain is lactobacillus plantarum Lactiplantibacillus plantarum (Gen Bank accession number is OQ 847791). J (J) ₁ The strain was Saccharomyces cerevisiae Meyerozyma guilliermondii, also Meng Maiye (GenBank accession number OQ 851829).

2. Sensory evaluation was performed on the fermented peppers. 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ The group has the phenomenon of incomplete fermentation and maturation, and the fermentation smell is light. 10 ¹⁰ :10 ⁹ The sourness is too strong and the brittleness is obviously reduced after group fermentation. 10 ⁹ :10 ⁸ The group sensory evaluation was best, 89.26 points.

3. And (5) measuring the pH value change of the low-salt fermented peppers with different bacteria concentrations. 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ The pH value of the fermented peppers in the group is obviously reduced (p is less than 0.05) to 4.19, 4.14,3.75 and 3.56 respectively, and the higher the bacterial concentration is, the lower the pH value is.

4. And measuring the total acid change of the low-salt fermented peppers with different bacteria concentrations. 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ The total acid of the group reaches 0.39g/100g, 0.48g/100g, 0.74g/100g and 0.82g/100g respectively, and the higher the bacterial concentration is, the faster the fermentation acid production rate is.

5. And measuring the reducing sugar change of the low-salt fermented peppers with different bacteria concentrations. 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ The reducing sugar content of the group was reduced to 2.10g/10, respectivelyThe higher the bacterial concentration, the more the reducing sugar utilization degree is, 0g, 2.05g/100g, 1.82g/100g and 1.54g/100 g.

6. And (5) measuring the amino acid nitrogen change of the low-salt fermented peppers with different bacteria concentrations. 10 ⁷ :10 ⁶ 、10 ⁸ :10 ⁷ 、10 ⁹ :10 ⁸ 、10 ¹⁰ :10 ⁹ The amino acid nitrogen content of the groups increased to 0.287g/100g, 0.291g/100g, 0.308g/100g, 0.316g/100g, respectively. As the concentration of bacteria increases, the protein availability of microorganisms to the pepper increases.

7. Determination of the organic acid content after fermentation, 10 ⁹ :10 ⁸ The highest organic acid content was 84.74g/kg.

8. Determination of volatile aroma substances in fermented Capsici fructus 10 ⁹ :10 ⁸ The most volatile aroma substances are 40.77% and 19.84% based on the highest ratio of esters to acids. 10 ⁹ :10 ⁸ The number of aroma substances having a relative odor activity value rOAVs of more than 0.1 is at most 36.

9. Obtained by cluster analysis and principal component analysis, 10 ⁹ :10 ⁸ The group contains main characteristic aroma substances such as nonanal (rovs= 224.12), linalool (rovs=40.96), hexanal (rovs=12.99), hexyl acetate (rovs=9.87), 4-undecalaldehyde (rovs=3.90), ethylphenol (rovs=3.59), and D-limonene (rovs=2.01).

In view of the fact that accurate control of bacterial liquid concentration in mass production is not easy to realize, lactobacillus plantarum R ₃ The concentration of the bacteria is 10 ⁹ About CFU/mL, meyer's patches J ₁ The concentration of the bacteria is 10 ⁸ When CFU/mL is about, the quality of the low-salt fermented pepper is optimal.

The invention screens out a strain of lactobacillus plantarum and Meng Maiye microzyme from natural fermented capsicum, is applied to low-salt fermented capsicum, adds the fragrance quality of the low-salt fermented capsicum, has low acidity generated by fermentation, can avoid the pollution of mixed bacteria in the low-salt fermented capsicum, prolongs the storage shelf life, and does not need to add preservative and flavor regulator.

The foregoing examples are set forth in order to provide a more thorough description of the present invention, and are not intended to limit the scope of the invention, since modifications of the present invention, in which equivalents thereof will occur to persons skilled in the art upon reading the present invention, are intended to fall within the scope of the invention as defined by the appended claims.

Claims (10)

1. A composite starter culture, comprising: lactobacillus plantarum bacterial liquidLactobacillus plantarumAnd Meng Maiye Saccharomyces cerevisiae liquidMeyerozyma guilliermondiiThe method comprises the steps of carrying out a first treatment on the surface of the The lactobacillus plantarumLactobacillus plantarumThe strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.27361, the preservation time is 2023, 5 and 17 days, and the preservation address is North Chenxi Lu No. 1, 3 in the Chaoyang area of Beijing city of China;

the Meng Maiye yeastMeyerozyma guilliermondiiThe microbial strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.27360, the preservation time is 2023, 5 and 17 days, and the preservation address is North Chenxi Lu No. 1, 3 in the Chaoyang area of Beijing city of China.

2. The composite starter according to claim 1, wherein the preparation method of the lactobacillus plantarum bacterial liquid comprises the following steps: picking single colony to culture medium, culturing at 37+ -5deg.C for 20-28 hr, and activating for several times; centrifuging, collecting thallus, washing with sterile physiological saline, and adjusting bacterial liquid concentration by Maillard turbidimetry;

The preparation method of the saccharomycete liquid comprises the following steps: selecting single colony to culture medium, culturing at 28+ -5deg.C for 20-28 hr, activating twice, centrifuging, collecting thallus, washing with sterile physiological saline, and regulating yeast amount by blood cell counting method.

3. The composite fermentation agent according to claim 1, wherein the viable count of lactobacillus plantarum in the lactobacillus plantarum bacterial liquid is 1 x 10 ⁹ ～3×10 ⁹ cfu/mL。

4. The composite fermentation agent according to claim 1, wherein the number of viable yeasts in the yeast liquid is 1×10 ⁸ ～3×10 ⁸ cfu/mL。

5. Use of a composite starter according to any one of claims 1-4 in the preparation of fermented peppers.

6. A fermented pepper, characterized in that the fermented pepper comprises the composite starter as claimed in any one of the claims 1-4.

7. A method of preparing fermented peppers, comprising: fermenting capsicum using the composite starter as claimed in any one of claims 1-4 to obtain a fermented capsicum.

8. The method of claim 7, wherein the concentration of the starter is 6-9%, and the strain ratio is 1:

(1-2)。

9. the method of claim 7, wherein the fermentation is a light-protected fermentation at 35 ℃ for 15-20 days.

10. The method of claim 7, wherein the pepper contains salt in an amount of 4% -8% (w/w).