A fermenting agent for fermenting salted pepper juice and a preparation method and application thereof

Abstract

The application provides a fermenting agent for fermented salted pepper juice, a preparation method and application thereof, and belongs to the technical field of food processing. The fermenting agent comprises Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664, and the total viable bacterial concentration is 7.34-9.17*10 10 CFU / mL ‑1 or 2.88-6.23*10 13 CFU / g. The application further provides a method for fermenting salted pepper juice, which comprises the following steps: S1) mixing salted pepper juice, fresh sauce, baijiu (Chinese liquor) and glucose to obtain a mixture; S2) inoculating the mixture with the fermenting agent, and sealing and fermenting to obtain a fermentation product; and S3) performing solid-liquid separation on the fermentation product to obtain flavor filter sauce and flavor filter liquid respectively. The flavor filter sauce and flavor filter liquid obtained by fermenting the salted pepper juice can be added back to pepper sauce and water juice chopped pepper products, so that the flavor and taste of the products can be efficiently and low-costly improved, and the product quality can be improved.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, and in particular relates to a fermenting agent for fermenting salted chili juice, its preparation method and application. Background Technology

[0002] Chopped chili peppers are a specialty of Hunan province and a key condiment in Hunan cuisine. Traditional chopped chili peppers are made by washing, drying, chopping, mixing with salt, and fermenting in a jar. They are characterized by a rich variety of fermentation bacteria, fully utilizing microbial resources, resulting in a mild flavor and a strong savory aroma. However, traditional chopped chili peppers require a long fermentation time, involve a complicated process, and have inconsistent fermentation quality. Currently, most chopped chili peppers are processed quickly by adding acid, and these quick-fermented products often lack proper fermentation and are essentially "quickly salted" chili peppers. "Quickly salted" chili peppers are made by draining the salted chili pepper extract from chili pepper embryos (preserved with a salt content of around 20%), rinsing with water to remove excess salt, and then adding acid and other additives. This process results in a low degree of fermentation, and the flavor is far inferior to traditionally fermented chopped chili peppers. The process also produces a large amount of salted chili pepper extract, and while rinsing removes excess salt, it also removes a significant amount of flavor and nutrients. The discharge of salted chili juice as waste liquid not only results in a huge waste of resources but also causes serious environmental pollution, severely hindering the high-quality and sustainable development of the fermented chili industry. Therefore, how to efficiently and cost-effectively enhance the flavor of industrially produced chopped chili peppers while avoiding environmental pollution and resource waste caused by rinsing and desalting the raw materials is a problem worthy of research. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a fermenting agent for fermenting salted chili juice, its preparation method and application; the fermenting agent provided by this invention can ferment salted chili juice, and the fermented product can efficiently and cost-effectively enhance aroma and flavor, and improve the product quality of chili sauce and chopped chili products.

[0004] This invention provides a starter culture for fermenting salted chili juice, comprising Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D, and Lactobacillus fermentum 664; the total viable bacterial concentration of the starter culture is 7.34–9.17 × 10⁻⁶. 10 CFU·mL -1 Or 2.88~6.23×10 13 CFU / g;

[0005] The ratio of viable counts of Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664 is (1-2):(1-2):(1-2).

[0006] Preferably, the preservation number of Pediococcus pentosaceus 6-12 is CGMCC No. 23496, the preservation number of Lactobacillus plantarum H3D is CGMCC No. 23495, and the preservation number of Lactobacillus fermentum 664 is CGMCC No. 25609.

[0007] This invention provides a method for preparing the aforementioned fermenting agent, comprising the following steps:

[0008] 1) After activating Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664 respectively, they were mixed to obtain a compound bacterial culture activation solution;

[0009] 2) The activated solution of the compound bacteria is inoculated into a high-density liquid culture medium for high-density fermentation culture to obtain fermentation broth.

[0010] Preferably, the high-density liquid culture medium, using water as a solvent, comprises the following components at the following concentrations: grape juice 50-100 g / L, L-glutamic acid 4-10 g / L, pyridoxal phosphate 0.05-0.1 mol / L, yeast extract 15-30 g / L, tryptone 10-20 g / L, MnSO4 concentration 0.45-0.55 g / L, sodium acetate 4.5-5.5 g / L, diammonium hydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, and Tween-80 0.8-1.2 g / L.

[0011] Preferably, the inoculation amount of the compound microbial activation solution is 0.8% to 1.2% of the mass of the high-density liquid culture medium, the high-density fermentation temperature is 30 to 37°C, the high-density fermentation time is 24 to 48 hours, and the pH value of the fermentation system is controlled at 6.0 to 6.5 during the high-density fermentation process.

[0012] This invention provides a method for fermenting salted chili juice using the aforementioned fermenting agent, comprising the following steps:

[0013] S1) Mix salted chili sauce, fresh sauce, white wine, and glucose to obtain a mixture;

[0014] S2) Inoculate the mixture with the fermenting agent and seal it for fermentation to obtain the fermentation product;

[0015] S3) The fermentation product is subjected to solid-liquid separation to obtain flavored sauce and flavored filtrate, respectively.

[0016] Preferably, the fresh sauce in step S1) includes chili sauce and tomato sauce; the mass ratio of the chili sauce and tomato sauce is (1-2):(1-2); the mass of the fresh sauce is 60%-100% of the mass of the salted chili juice;

[0017] The alcohol content of the liquor is 45% to 55%, and the mass of the liquor is 1% to 4% of the mass of the salted chili juice; the mass of the glucose is 2% to 6% of the mass of the salted chili juice.

[0018] Preferably, the inoculum amount of the fermenting agent in step S2) is 0.1% to 2% of the mass of the mixture, and the sealed fermentation time is 5 to 10 days.

[0019] The present invention provides flavored sauces or flavored filtrates prepared by the method described above.

[0020] This invention provides the application of the flavored filter sauce or flavored filtrate in flavor-enhancing chopped chili products, wherein the flavored filter sauce is used in flavor-enhancing chili sauce products, and the flavored filtrate is used in flavor-enhancing water-based chopped chili products.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The fermentation agent provided by the present invention is obtained by high-density fermentation culture of Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664, resulting in a high concentration of live bacteria and a high survival rate of cells after freeze-drying; When the fermentation agent of the present invention is used to ferment salted chili juice, the total organic acid content in the product is appropriate, mainly lactic acid, citric acid and malic acid, and the organic acid structure is reasonable; The content of amino acid nitrogen, free amino acids and γ-aminobutyric acid in the product is high; At the same time, the content of linoleic acid, α-linolenic acid and arachidonic acid is also increased; And the fermentation product has significant characteristic aroma substances.

[0022] Furthermore, this invention involves adding the flavored filter sauce and flavored filtrate obtained from the fermentation of salted chili juice back into the chili sauce and water-based chopped chili products, respectively. This efficiently and cost-effectively enhances aroma and flavor, improving product quality. The chili sauce prepared by this invention exhibits higher sensory scores, amino acid nitrogen content, total free amino acid content, and total aroma substance content than industrially produced chili sauces. Similarly, the water-based chopped chili prepared by this invention also shows higher sensory scores, amino acid nitrogen content, total free amino acid content, and total aroma substance content than industrially produced water-based chopped chili. γ-aminobutyric acid (GABA) was detected in both the chili sauce and the water-based chopped chili of this invention, while GABA was not detected in either industrially produced chili sauce or industrially produced water-based chopped chili. Attached Figure Description

[0023] Figure 1 The processing flow of the experimental group in Experiment Example 1;

[0024] Figure 2 The processing flow of control group 1 in Experiment Example 1;

[0025] Figure 3 The processing flow of control group 2 in Experiment Example 1;

[0026] Figure 4 The processing flow of control group 3 in Experiment Example 1;

[0027] Figure 5 The processing flow is for control group 4 in Experiment Example 1;

[0028] Figure 6 The process flow for processing chili sauce and chopped chili in water according to the present invention is shown in Experimental Example 2;

[0029] Figure 7 The processing flow of existing chili sauce and chopped chili sauce in Experiment Example 2 is shown.

[0030] Biological Preservation Instructions

[0031] Pediococcus pentosaceus 6-12, accession number: CGMCC NO.23496, deposited at China General Microbiological Culture Collection Center, deposited on September 28, 2021, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0032] Lactobacillus plantarum H3D, accession number: CGMCC NO.23495, deposited at China General Microbiological Culture Collection Center, on September 28, 2021, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0033] Lactobacillus fermentum 664, accession number: CGMCC NO.25609, deposited at China General Microbiological Culture Collection Center, on August 29, 2022, at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0034] This invention provides a starter culture for fermenting salted chili juice, comprising Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D, and Lactobacillus fermentum 664; the total viable bacterial concentration of the starter culture is 7.34–9.17 × 10⁻⁶. 10 CFU·mL -1 Or 2.88~6.23×10 13 CFU / g; the ratio of viable counts of Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664 is (1-2):(1-2):(1-2).

[0035] In this invention, the preservation number of *Pediococcus pentosaceus* 6-12 is CGMCC No. 23496, the preservation number of *Lactobacillus plantarum* H3D is CGMCC No. 23495, and the preservation number of *Lactobacillus fermentum* 664 is CGMCC No. 25609. In this invention, the starter culture is preferably a solid powder.

[0036] The present invention provides a method for preparing the aforementioned fermentation agent, comprising the following steps: 1) activating Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664 and mixing them to obtain a compound bacterial culture activation solution; 2) inoculating the compound bacterial culture activation solution into a high-density liquid culture medium for high-density fermentation culture to obtain a fermentation broth.

[0037] In this invention, *Pediococcus pentosaceus* 6-12, *Lactobacillus plantarum* H3D, and *Lactobacillus fermentum* 664 are activated and then mixed to obtain a composite bacterial culture activation solution. Preferably, the preserved *Pediococcus pentosaceus* 6-12, *Lactobacillus plantarum* H3D, and *Lactobacillus fermentum* 664 bacterial suspensions are inoculated separately into a high-density culture medium for activation culture. The activation culture temperature is preferably 36–38°C, and the activation culture time is preferably 12–24 hours. After the activation culture is completed, the step of adjusting the bacterial suspension concentration is preferably included, more preferably using sterile physiological saline. The concentration of the bacterial suspension is preferably adjusted to 0.4–0.6 McFarland turbidity tubes. Then, the three bacterial suspensions are mixed, and the volume ratio of the mixture is preferably 1:1:1.

[0038] In this invention, the high-density liquid culture medium, using water as a solvent, preferably comprises the following components at the following concentrations: grape juice 50-100 g / L, L-glutamic acid 4-10 g / L, pyridoxal phosphate 0.05-0.1 mol / L, yeast extract 15-30 g / L, tryptone 10-20 g / L, MnSO4 concentration 0.45-0.55 g / L, sodium acetate 4.5-5.5 g / L, diammonium hydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 1-3 g / L, 0. The concentration of Tween-80 is 8–1.2 g / L, and more preferably, it includes the following components at the following concentrations: 60–90 g / L of grape juice, 5–9 g / L of L-glutamic acid, 0.06–0.09 mol / L of pyridoxal phosphate, 18–28 g / L of yeast extract, 12–18 g / L of tryptone, 0.5 g / L of MnSO4, 5.0 g / L of sodium acetate, 1.5–2.5 g / L of diammonium hydrogen phosphate, 1.5–2.5 g / L of dipotassium hydrogen phosphate, and 1.0 g / L of Tween-80. In this invention, the grape juice is the juice obtained after washing grapes and then crushing and filtering them using a tissue homogenizer.

[0039] After obtaining the activated compound microbial culture solution, this invention inoculates the activated compound microbial culture solution into a high-density liquid culture medium for high-density fermentation to obtain a fermentation broth. In this invention, the inoculation amount of the activated compound microbial culture solution is preferably 0.8%–1.2% of the mass of the high-density liquid culture medium, more preferably 0.9%–1.1%; the high-density fermentation temperature is preferably 30–37°C, more preferably 35–37°C; the high-density fermentation time is preferably 24–48 hours, more preferably 32–40 hours; in this invention, the high-density fermentation is carried out in a fermenter, and during the high-density fermentation process, stirring is performed, with the stirring speed preferably 150–250 r / min, more preferably 200 r / min. In this invention, the pH value of the fermentation system is controlled at 6.0–6.5 during the high-density fermentation process. This invention preferably uses 15–20% (m / m) ammonia water as a neutralizing agent to control the pH value of the fermentation system. After obtaining the fermentation broth, the present invention preferably further includes the following steps: solid-liquid separation of the fermentation broth, collection of bacterial sludge, mixing of the bacterial sludge with a freeze-drying protectant, and freeze-drying to obtain fermentation agent powder. In the present invention, the solid-liquid separation is preferably performed by centrifugation at a temperature of 3–5°C, a rotation speed of 5000–8000 r / min, and a centrifugation time of 6–10 min. In the present invention, the freeze-drying protectant is preferably water-based and comprises the following components at concentrations: 6–10 wt% skim milk powder, 6–12 wt% xylooligosaccharides, 50–100 mg / L vitamin B6, and 1–2 wt% L-glutamic acid. In the present invention, the volume of the freeze-drying protectant added is preferably the same as the volume of the supernatant collected after centrifugation of the fermentation broth. In this invention, pre-freezing is preferably performed before freeze-drying; pre-freezing is preferably performed at -60℃ to -85℃ for 2 to 6 hours; freeze-drying is preferably performed in a freeze dryer, and the freeze-drying parameters are preferably: vacuum degree 0.005 to 0.25 Pa, temperature -90℃ to -105℃, and time 20 to 50 hours.

[0040] The present invention provides a method for fermenting salted chili juice using the aforementioned fermenting agent, comprising the following steps: S1) mixing salted chili juice, fresh sauce, white wine, and glucose to obtain a mixture; S2) inoculating the mixture with the fermenting agent and sealing it for fermentation to obtain a fermentation product; S3) separating the fermentation product into solid and liquid components to obtain flavored sauce and flavored filtrate, respectively.

[0041] In this invention, a mixture is obtained by mixing pickled chili juice, fresh sauce, liquor, and glucose. The fresh sauce preferably includes chili sauce and tomato sauce; the mass ratio of the chili sauce to tomato sauce is preferably (1-2):(1-2), more preferably 1:1; both the chili sauce and tomato sauce are preferably freshly made; the mass of the fresh sauce is preferably 60%-100% of the mass of the pickled chili juice; the alcohol content of the liquor is preferably 45%-55%, and the mass of the liquor is preferably 1%-4% of the mass of the pickled chili juice, more preferably 2%-3%; the mass of the glucose is preferably 2%-6% of the mass of the pickled chili juice, more preferably 3%-5%. The purpose of mixing pickled chili juice, fresh sauce, liquor, and glucose in this invention is threefold: first, to reduce salt content; second, to provide nutritional support for the fermentation agent combination of this invention; and third, to use liquor to inhibit the growth of unwanted microorganisms and enhance flavor.

[0042] In this invention, after obtaining the mixture, the fermenting agent is inoculated into the mixture, and fermentation is carried out in a sealed manner to obtain the fermentation product. In this invention, the inoculation amount of the fermenting agent is preferably 0.1% to 2% of the mass of the mixture, more preferably 0.5% to 1.5%; in this invention, the sealed fermentation is preferably carried out in a fermentation jar, with the above-mentioned fermentation raw materials placed in the jar, and the distance between the raw materials and the jar opening is preferably 3 to 10 cm; in this invention, the jar is preferably covered with a lid, and water is added to the lid to seal it; in this invention, the sealed fermentation time is preferably 5 to 10 days.

[0043] After obtaining the fermentation product, the present invention performs solid-liquid separation to obtain flavored sauce and flavored filtrate, respectively. In this invention, the solid-liquid separation is preferably performed by centrifugation, with a centrifugation speed preferably of 4000–6000 r / min, more preferably 4500–5500 r / min, and most preferably 5000 r / min; the centrifugation time is preferably 5–10 min, more preferably 6–9 min.

[0044] The present invention also provides flavored sauces or flavored filtrates prepared by the method.

[0045] This invention provides the application of the flavored filter sauce or flavored filtrate in flavor-enhancing chopped chili products, wherein the flavored filter sauce is used in flavor-enhancing chili sauce products, and the flavored filtrate is used in flavor-enhancing water-based chopped chili products.

[0046] In this invention, the chili sauce product preferably comprises the following components: 60-80% salted chili sauce, 3-10% flavored filter sauce, 3-5% tomato sauce, 3-5% chili powder, 3-4% black tiger freeze-dried powder, 0.5-1% monosodium glutamate, 3-5% minced garlic, and 4.5-10% edible vegetable oil.

[0047] In this invention, the water-based chopped chili product preferably comprises the following components: 60-80% solid salted chili peppers, 17-37% flavor filtrate, 0.5-1% monosodium glutamate, and 0.5-2% garlic cloves.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] (1) Activation with 6-12, H3D, and 664

[0051] Take out the 6-12, H3D, and 664 glycerol preservation tubes stored at -80℃, thaw them at 4℃, take 1mL of the preserved bacterial suspension and incubate it in 100mL of high-density liquid culture medium at 37℃ for 24h, adjust the bacterial suspension concentration to 0.5 McFarland turbidity tubes with sterile physiological saline, and mix the bacterial suspensions of 6-12, H3D, and 664 in a volume ratio of 1:1:1 to prepare the compound bacterial activation solution.

[0052] (2) High-density culture

[0053] Based on the weight of the high-density liquid culture medium, the compound bacterial culture activation solution was inoculated into the fermenter at an inoculation rate of 1% (m / m). The mixture was stirred at 200 rpm, and 20% (m / m) ammonia was used as a neutralizing agent to control the pH of the fermentation system between 6.0 and 6.5. Fermentation and enrichment culture was carried out at 37℃ for 24 h to obtain the fermentation broth. 1 mL of the fermentation broth was taken and serially diluted with 0.9% (m / m) physiological saline, spread on plates, and viable cell counts were performed. The fermentation broth was centrifuged at 8000 rpm at 4℃ for 10 min to remove the supernatant. The precipitate was washed with sterile physiological saline, centrifuged again, and the supernatant was removed. This washing process was repeated twice to obtain the bacterial sludge.

[0054] (3) Vacuum freeze drying

[0055] Add an equal volume of freeze-drying protectant to the bacterial sludge, mix well, and pre-freeze at -85℃ for 6 hours. Finally, place the frozen material into a freeze dryer for freeze-drying. The freeze dryer parameters are set as follows: vacuum degree 0.25 Pa, temperature -105℃, and drying time 50 hours. After obtaining the freeze-dried bacterial powder, seal and store it in a refrigerator at 4℃. Take 1g of the bacterial powder, perform serial dilutions with 0.9% physiological saline, and spread it on plates for pre-freeze-drying viable cell count.

[0056] The high-density liquid culture medium uses water as a solvent and has the following composition: 100 g / L grape juice, 10 g / L L-glutamic acid, 0.1 mol / L pyridoxal phosphate, 30 g / L yeast extract, 20 g / L tryptone, 0.50 g / L MnSO4, 5 g / L sodium acetate, 1-3 g / L diammonium hydrogen phosphate, 3 g / L dipotassium hydrogen phosphate, and 1 g / L Tween-80.

[0057] The grape juice is the juice obtained by washing grapes, crushing and filtering them using a tissue homogenizer.

[0058] The freeze-drying protectant is composed of the following components: 10% skim milk powder, 12% xylooligosaccharide, 100 mg / L vitamin B6, and 2% L-glutamic acid, based on the weight of water (m / m).

[0059] Survival rate calculation:

[0060] After counting the viable bacteria in the freeze-dried powder, the survival rate of lactic acid bacteria is calculated using the following formula:

[0061] Lactic acid bacteria survival rate = N 1* W1 / N 0* W0×100%

[0062] In the formula: N1 represents the number of viable bacteria per 1g of freeze-dried bacterial powder (CFU / g).

[0063] N0 represents the number of viable bacteria per 1g of liquid sample before freeze-drying (CFU / g).

[0064] W1 represents the weight (g) of the bacterial powder obtained after freeze-drying.

[0065] W0 represents the total weight (g) of the bacterial culture before freeze-drying.

[0066] After high-density cultivation, the viable cell concentration in the fermentation broth reached 7.34–9.17 × 10⁻⁶. 10 CFU·mL -1 After vacuum freeze-drying, the bacterial cell survival rate was 96.14%–98.21%, and the viable bacterial count in the powder was 2.88–6.23 × 10⁻⁶. 13 CFU / g.

[0067] Comparative Example 1

[0068] Preparation of fermentation agent

[0069] The difference from Example 1 is that step (2) is cultured in ordinary MRS medium, and the formulation of the freeze-drying protectant in step (3) is: based on the weight of water (m / m), 10% skim milk powder, 12% xylooligosaccharide, and 2% sodium L-glutamate, with other conditions being the same.

[0070] After culturing in ordinary MRS medium, the viable cell concentration in the fermentation broth was 2.02–6.15 × 10⁻⁶. 9 CFU·mL -1 After vacuum freeze-drying, the bacterial cell survival rate was 94.22%–96.58%, and the viable bacterial count in the powder was 3.01–4.55 × 10⁻⁶. 11 CFU / g.

[0071] Experimental Example 1

[0072] (I) Experimental Grouping

[0073] Experimental group: Inoculated with 0.2% of the starter culture from Example 1;

[0074] Control group 1: No inoculation, natural fermentation;

[0075] Control group 2: Inoculated with 0.2% of the starter culture (starter culture prepared by the method of Comparative Example 1);

[0076] Control group 3: Inoculated with 0.2% of starter culture A, in which the ratio of *Lactobacillus plantarum*: *Lactobacillus brevis*: *Westernella enteroides* was 2:1:1, with a concentration of 2.88–6.23 × 10⁻⁶. 13 CFU / g;

[0077] Control group 4: Inoculated with 0.2% of the fermentation agent described in Example 2 of CN117683681B.

[0078] Salted chili juice desalination and inoculation fermentation process:

[0079] The salt reduction method is as follows: add 80% fresh sauce (fresh chili sauce: tomato sauce = 1:1) to the salted chili sauce; add 3% white wine (45% alcohol content) to the salted chili sauce; and add 4% glucose to the salted chili sauce.

[0080] Inoculation with fermenting agent: 0.1% of the total mass of all raw materials after salt reduction.

[0081] Filling the jars: Fill the jars with the above materials, leaving a 10cm gap from the rim.

[0082] Seal: Cover the jar with the lid, add water to seal, and ferment for 10 days.

[0083] Centrifugal filtration: Centrifuge speed 5000r / min, centrifugation time 5-10min to separate solid and liquid, to obtain flavored sauce and flavored filtrate. The flavored residue is used to enhance the aroma and flavor of chili sauce products, and the flavored filtrate is used to enhance the aroma and flavor of chopped chili sauce products.

[0084] (II) Composition and content of organic acids in fermentation broth

[0085] Sample preparation: Accurately weigh 2.50 g of fermentation broth sample and place it in a 50 mL volumetric flask. Extract in a 75 °C water bath for 20 min, cool to room temperature, make up to 50 mL, filter, centrifuge the filtrate (4000 r / min, 20 min), filter the supernatant through a 0.45 μm microporous membrane, and inject for analysis.

[0086] The determination of organic acids was carried out in accordance with the national food safety standard GB5009.157-2016, "Determination of Organic Acids in Food".

[0087] The organic acid content of the experimental group and the control group is shown in Table 1.

[0088] Table 1. Organic acid content (mg / g) in the experimental and control groups.

[0089] organic acids experimental group Control group 1 Control group 2 Control group 3 Control group 4 lactic acid 3.0897 1.8078 3.4454 2.3378 2.9656 Citric acid 1.6554 1.4312 1.3927 1.6614 1.4623 malic acid 2.6415 1.4578 2.2629 2.7814 2.6101 oxalic acid 0.4736 0.2701 0.2524 3.9847 3.8201 Acetic acid 0.5456 0.5234 0.5178 0.5996 0.5956 tartaric acid 1.5023 2.4765 1.3525 3.9032 3.7765 propionic acid Not detected 0.1357 Not detected 0.01978 Not detected butyric acid Not detected 0.1214 Not detected 0.04587 Not detected

[0090] As shown in Table 1, the total organic acid contents of the experimental group, control group 1, control group 2, control group 3, and control group 4 were 9.9081 mg / g, 8.2239 mg / g, 9.2237 mg / g, 15.33375 mg / g, and 15.2302 mg / g, respectively. This indicates that the total organic acid content of the experimental group was relatively appropriate, with lactic acid, citric acid, and malic acid being the main organic acids, and the organic acid structure was reasonable.

[0091] Propionic acid and butyric acid are both putrefactive acids produced by putrefactive bacteria in fermented vegetables. They not only consume nutrients in the vegetables but also cause spoilage and a foul odor. Therefore, it is essential to control the microbial community structure of fermented vegetables, minimizing the presence of butyric acid bacteria, propionic acid bacteria, and carboxylic acid bacteria to reduce or avoid the production of putrefactive acids such as propionic acid and butyric acid. Korean food hygiene standards stipulate that propionic acid must not be detected in pickled vegetables (detection limit 30 mg / kg). The fermenting agent of this invention, by strengthening the structure and dosage of lactic acid bacteria, optimizes the microbial community structure of fermented chili juice, thus avoiding the detection of propionic and butyric acids.

[0092] (III) Study on amino acid nitrogen content of fermentation broth

[0093] The amino acid nitrogen content of the experimental group and the control group was determined according to GB 5009.235-2016, and the results are shown in Table 2.

[0094] Table 2 Comparison of Amino Acid Nitrogen Content

[0095]

[0096] The results showed that after 7 days of fermentation, the amino acid nitrogen content of the starter culture of Example 1 (experimental group), prepared by culture in high-density liquid medium according to the present invention, reached 0.32 g / 100 g, followed by the starter culture of Example 2 (control group 2), prepared by culture in ordinary MRS medium, with an amino acid nitrogen content of 0.31 g / 100 g. In contrast, the amino acid nitrogen content of the natural fermentation group (control group 1) was only 0.20 g / 100 g, and the amino acid nitrogen contents of control groups 3 and 4 were only 0.25 g / 100 g and 0.30 g / 100 g, respectively. This indicates that inoculation with the starter culture of the present invention can increase the amino acid nitrogen content of the fermentation broth.

[0097] (iv) Determination of free amino acid content in fermentation broth

[0098] Sample pretreatment: Weigh 1.000 g of each of the inoculated fermentation broth (experimental group), control group 1, control group 2, control group 3, and control group 4 into a 10 mL centrifuge tube. Add 5 mL of 0.01 mol / L hydrochloric acid, mix well, boil in a water bath for 15 min, sonicate for 10 min, centrifuge at 4000 rpm for 5 min, and collect the supernatant. Resuspend the precipitate in 4 mL of 0.01 mol / L hydrochloric acid, sonicate for 10 min, centrifuge, combine the supernatants, and bring the volume to 10 mL. Perform membrane analysis.

[0099] Online pre-column derivatization: An automated online derivatization method from Agilent Technologies was used. Primary amino acids were derivatized with o-phthalaldehyde (OPA), and secondary amino acids with fluorenemethyloxycarbonyl chloride (FMOC), followed by column chromatography and detection. Chromatographic conditions: ZORBAX EclipseAAA (4.6 x 75 mm, 3.5 μm); Detection signal: UV 338 nm (0–19 min), 266 nm (19.01–25 min); Mobile phase A: 40 mmol / L sodium dihydrogen phosphate (pH 7.8); Mobile phase B: methanol / acetonitrile / water = 45 / 45 / 10; Flow rate: 1.0 mL / min.

[0100] Gradient elution process: 100% A: 0% B (0-1 min): 46% A; 57% B (23 min): 0% A: 100% B (27-34 min); 100% A: 0% B (40-41 min).

[0101] The contents of each free amino acid in the experimental group and the control group are shown in Table 3.

[0102] Table 3. Content of each free amino acid in the experimental group and the control group (mg / g)

[0103]

[0104]

[0105] As shown in Table 3, the experimental group had the highest content of 17 free amino acids in its fermentation broth, reaching 7.0172 mg / g, followed by control group 2 at 6.5015 mg / g. The total content of 17 free amino acids in control group 1 was only 2.6896 mg / g, while the contents of 17 free amino acids in the fermentation broths of control groups 3 and 4 were only 5.2753 mg / g and 5.83 mg / g, respectively. Therefore, this indicates that the fermenting agent prepared by culturing the microbial combination of the present invention in a high-density liquid culture medium (i.e., the fermenting agent of Example 1) is suitable for fermenting salted chili juice and has the ability to increase the content of free amino acids in the fermentation broth.

[0106] (V) Determination of γ-aminobutyric acid content

[0107] Gamma-aminobutyric acid (GABA) is a non-protein amino acid and an important inhibitory neurotransmitter in the central nervous system. Approximately 30% of central nervous system synapses use GABA as a neurotransmitter. When the body is deficient in GABA, it can lead to anxiety, restlessness, fatigue, and worry.

[0108] The content of γ-aminobutyric acid was determined by HPLC.

[0109] (1) Preparation of standard stock solution of γ-aminobutyric acid

[0110] Accurately weigh 10.0 mg of γ-aminobutyric acid (GABA) standard, dissolve it in acetonitrile solution and dilute to 10 mL to obtain a 100 mg / L standard stock solution. Store the stock solution in a sealed brown glass bottle at -18°C. When using, serially dilute the standard stock solution to prepare γ-aminobutyric acid standard working solutions of 2.0 mg / L, 5.0 mg / L, 10.0 mg / L, 50.0 mg / L and 100.0 mg / L or other concentrations. Prepare fresh solutions immediately before use.

[0111] (2) Sample preparation

[0112] The test strain, previously preserved in glycerol, was inoculated into MRS liquid medium for three generations of activation. Then, it was inoculated into 8 mL of TYG fermentation medium at a 4% (V / V) inoculation ratio and cultured at 37°C for 48 h. After thorough mixing, the cultured strain was centrifuged in a 10 mL centrifuge tube (8000 rpm for 10 min), and the supernatant was collected for later use.

[0113] (3) Extraction

[0114] Weigh 1.0 g of fermentation supernatant into a 50 mL centrifuge tube, add 10 mL of extraction solution, sonicate for 30 min, vortex for 2 min, let stand for 5 min, centrifuge at 5000 r / min for 5 min, transfer the supernatant to a 25 mL volumetric flask, extract the fermentation broth residue once more with 10 mL of extraction solution, combine the two extracts, bring the volume to 25 mL with the extraction solution, shake well, and allow for derivatization.

[0115] (4) Sample derivatization reaction

[0116] Accurately pipette 1 mL of the above-mentioned diluted sample solution into a stoppered test tube, add 0.20 mL of sodium bicarbonate solution and 0.40 mL of 4-dimethylaminoazobenzene-4-sulfonyl chloride derivatizing reagent, mix well, and derivatize in a 70℃ water bath for 20 min. Filter with a microporous membrane and test.

[0117] (5) Chromatographic reference conditions for determination:

[0118] Chromatographic column: C18 column, 250mm x 4.6mm, 5µm;

[0119] Detection wavelength: 436nm;

[0120] Column temperature: 30℃;

[0121] Injection volume: 10 μL;

[0122] Mobile phase: acetonitrile + sodium acetate trihydrate solution (35+65); flow rate: 1.0 mL / min.

[0123] The γ-aminobutyric acid (GABA) standard curve was plotted using high performance liquid chromatography (HPLC) with the concentration of GABA as the x-axis and the peak area as the y-axis.

[0124] The γ-aminobutyric acid content in the experimental group and the control group is shown in Table 4.

[0125] Table 4. γ-aminobutyric acid (GABA) content in the experimental and control groups (g / L)

[0126]

[0127] The results showed that after 7 days of fermentation, the content of γ-aminobutyric acid (GABA) in the fermentation broth of the experimental group was 0.87 g / L, the content of GABA in the fermentation broth of control group 2 was 0.65 g / L, and GABA was not detected in other control groups. This indicates that the fermentation agent of the present invention can produce GABA, and the high-density culture medium of the present invention helps to improve the ability of the fermentation agent of the present invention to produce GABA.

[0128] (vi) Detection of fatty acids

[0129] Linoleic acid was determined according to the method of NY / T 3299-2018, α-linolenic acid was determined according to the method of GB 28404-2012, and arachidonic acid was determined according to the method of SN / T 2922-2022.

[0130] The contents of linoleic acid, α-linolenic acid and arachidonic acid in the experimental group and the control group are shown in Table 5.

[0131] Table 5. Fatty acid content (mg / L) in the experimental and control groups.

[0132]

[0133]

[0134] The results showed that after 7 days of fermentation, the contents of linoleic acid, α-linolenic acid, and arachidonic acid in the fermentation broth of the experimental group were 23.63 mg / L, 11.82 mg / L, and 16.32 mg / L, respectively. The contents of linoleic acid, α-linolenic acid, and arachidonic acid in the fermentation broth of control group 2 were 20.51 mg / L, 9.74 mg / L, and 15.36 mg / L, respectively. Linoleic acid, α-linolenic acid, and arachidonic acid were not detected in the other control groups. This indicates that the fermentation agent of the present invention can produce linoleic acid, α-linolenic acid, and arachidonic acid, and the high-density culture medium of the present invention helps to improve the ability of the fermentation agent of the present invention to produce linoleic acid, α-linolenic acid, and arachidonic acid.

[0135] (vii) Determination of aroma substance content in fermentation broth

[0136] The extraction head was aged at 270℃ for 0.5h.

[0137] Solid-phase microextraction: Take 2g of each of the experimental group, control group 1, control group 2, control group 3, and control group 4 into a 20mL headspace vial, add 3mL of water, place a small magnetic force, and quickly tighten the cap with the silicone rubber septum. After preheating at 70℃ for 15min on a constant temperature magnetic stirrer, insert the aged extraction head into the headspace vial and then push the extraction head out to a position about 0.7cm above the liquid surface in the headspace for adsorption for 40min; then inject and desorb for 2min.

[0138] Chromatographic conditions: Column: RTX-5ms flexible quartz capillary column (30m × 0.25mm × 0.25μm); Carrier gas: High-purity He (99.999%), flow rate: 1.0mL / min; Injector temperature: 250℃; Split injection. Temperature program: Column temperature 50℃, ramped at 4℃ / min to 180℃, held for 4 min, ramped at 6℃ to 250℃, held for 1 min.

[0139] Mass spectrometry conditions: Ion source: EI source; GC / MS interface temperature: 220°C; Ion source temperature: 200°C; Electron energy: 70 eV; Mass scan range: 35–500 amu.

[0140] Retention Index (RI): Based on the method described above, C7 to C6... 40 A mixture of n-alkanes as a reference standard was analyzed by GC-MS. The retention times of each n-alkanes were recorded, and the RI (residue ratio) of each component was calculated according to the program. Calculation formula: t x The retention time of the elution peak for the analyzed group; t n and t n+1 tn is the retention time (min) of the elution peak of n-alkanes with carbon atoms n and n+1, and tn < tx < tn+1.

[0141] Qualitative analysis: Qualitative analysis was performed by comparing the pure mass spectra of each component with the NIST2017 standard library, and the retention index (RI) values ​​were compared. The percentage content of each component was determined by internal standard method using compounds with similarity >80% and the closest retention index (RI) values.

[0142] Quantitative analysis: The internal standard method was used to quantitatively analyze the volatile substances in the fermentation broth, with o-dichlorobenzene as the internal standard. The content of volatile substances was calculated as follows.

[0143] Calculation formula:

[0144] In the formula: C j —Content of flavor compounds to be tested, μg / kg;

[0145] A j —The chromatographic peak area corresponding to the analyte in the sample;

[0146] A i —Determine the peak area of ​​the chromatographic sample using an internal standard;

[0147] V—Volume of internal standard, μL;

[0148] C i —Internal standard concentration, μg / mL;

[0149] M j —Sample mass, g.

[0150] The contents of isothiocyanate compounds and other volatile aroma substances in the fermentation broth of the experimental group and each control group are shown in Table 6.

[0151] Table 6. Content of characteristic aroma substances in the experimental group and each control group (μg / kg)

[0152]

[0153]

[0154]

[0155] Chili peppers produce a wide variety of aroma compounds after fermentation, numbering around 1000. Table 6 shows the characteristic flavor compounds and their content ranges that significantly influence the fermentation flavor. After 7 days of fermentation, the experimental group's fermentation broth contained all the characteristic aroma compounds listed in the standard, and the content of each compound was within the standard range. The total characteristic aroma compound content was the highest, reaching 2334.612 μg / kg. In contrast, the control group's fermentation broth did not contain all the characteristic aroma compounds listed in the standard, and the content of most compounds was outside the standard range. The control group 1 had the lowest total characteristic aroma compound content, at only 816.8075 μg / kg. Therefore, this demonstrates that the fermenting agent prepared by the microbial combination of the present invention through high-density liquid culture medium (i.e., the fermenting agent of Example 1) has a significant ability to produce characteristic aroma compounds when fermenting salted chili pepper juice.

[0156] Experimental Example 2

[0157] Chili sauce and broth chopped chili processing:

[0158] The processing flow of chili sauce and chopped chili sauce of this invention is as follows: Figure 6 As shown.

[0159] Sauce making: Salted chili sauce is obtained by grinding solid salted chili peppers into a fine powder using a colloid mill.

[0160] Formula 1: 60% salted chili sauce, 15% flavored strainer sauce, 5% tomato sauce, 3% chili powder, 1% MSG, 5% minced garlic, 10% vegetable oil.

[0161] Formula 2: 75% solid salted chili peppers, 22% flavor filtrate, 1% MSG, and 2% garlic cloves.

[0162] The existing processing flow for chili sauce and chopped chili sauce is as follows: Figure 7 As shown.

[0163] Rinse with water: Add water in a ratio of 1:1 (salted chili peppers: tap water) to rinse, filter, and desalinate to obtain rinsed and desalted chili peppers.

[0164] Sauce making: Desalted chili peppers are obtained by rinsing and desalting them and then grinding them finely with a colloid mill.

[0165] Formula 3: 75% desalted chili sauce, 5% tomato sauce, 4% chili powder, 1% MSG, 5% minced garlic, 10% vegetable oil.

[0166] Formula 4: 75% rinsed and desalted chili peppers, 22% sterile water, 1% MSG, and 2% garlic cloves.

[0167] Table 7 Sensory Evaluation Table for Chili Sauce

[0168]

[0169] Table 8 Sensory Evaluation Table for Chopped Chili Sauce

[0170]

[0171]

[0172] Table 9 Sensory scores and flavor nutrient content of the experimental and control groups.

[0173]

[0174] Existing processing techniques require rinsing and desalting. In the process of rinsing away excess salt, the nutrients and flavor substances in the salted peppers are also washed away, generating a large amount of brine that is discharged into the environment. This not only wastes water resources and nutrients and flavor substances, but also pollutes the environment. More importantly, the product has a poor flavor.

[0175] Using the method of this invention, the processing of chili sauce and chopped chili sauce with broth does not require rinsing and desalination, saving water and reducing waste and environmental pollution. It achieves zero discharge of brine during the processing of chili sauce and chopped chili sauce products. More importantly, the fermented chili juice is added back to the chili sauce and chopped chili sauce products, efficiently and cost-effectively enhancing aroma and flavor, and improving product quality. The sensory score, amino acid nitrogen content, total free amino acid content, and total aroma substance content of the chili sauce of this invention are 95.33 points, 0.32 g / 100g, 7.3372 mg / g, and 3324.2315 μg / kg, respectively, all higher than the contents of each indicator in industrially produced chili sauce. The sensory score, amino acid nitrogen content, total free amino acid content, and total aroma substance content of the chopped chili sauce with broth of this invention are 96.36 points, 0.31 g / 100g, 6.0258 mg / g, and 2659.3658 μg / kg, respectively, all higher than the contents of each indicator in industrially produced chopped chili sauce with broth. γ-aminobutyric acid (GABA) was detected in both the chili sauce and the chopped chili sauce of this invention, with contents of 0.46 g / kg and 0.34 g / kg, respectively. However, GABA was not detected in industrially produced chili sauce and industrially produced chopped chili sauce.

[0176] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fermenting agent for fermenting salted pepper juice, characterized by, Pediococcus pentosaceus (Pediococcus pentosaceus) Pediococcus pentosaceus Lactobacillus plantarum (Lactobacillus plantarum) Lactobacillus plantarum H3D and Lactobacillus fermentum (Lactobacillus fermentum) Lactobacillus fermentum 664; the total viable bacterial concentration of the starter culture is 7.34~9.17 ×10 10 CFU·mL -1 or 2.88~6.23×10 13 CFU / g; The starter is prepared by mixing the bacterial suspensions of Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664 in a volume ratio of 1:1:

1. The preservation number of Pediococcus pentosaceus 6-12 is CGMCC No. 23496, the preservation number of Lactobacillus plantarum H3D is CGMCC No. 23495, and the preservation number of Lactobacillus fermentum 664 is CGMCC No. 25609.

2. Process for the preparation of the starter culture according to claim 1, characterized in that, The method comprises the following steps: 1) obtaining a complex bacterial strain activation solution by mixing Pediococcus pentosaceus 6-12, Lactobacillus plantarum H3D and Lactobacillus fermentum 664 after being activated respectively; 2) obtaining a fermentation liquor by inoculating the complex bacterial strain activation solution in a high-density liquid culture medium for high-density fermentation culture. The high-density liquid culture medium, with water as a solvent, is composed of the following components in the following concentrations: 50-100 g / L of Vitis coignettii juice, 4-10 g / L of L-glutamic acid, 0.05-0.1 mol / L of pyridoxal phosphate, 15-30 g / L of yeast extract, 10-20 g / L of tryptone, 0.45-0.55 g / L of MnSO4, 4.5-5.5 g / L of sodium acetate, 1-3 g / L of diammonium hydrogen phosphate, 1-3 g / L of dipotassium hydrogen phosphate, and 0.8-1.2 g / L of Tween-80.

3. The method of claim 2, wherein, The inoculation amount of the complex bacterial strain activation solution is 0.8%-1.2% of the mass of the high-density liquid culture medium, the temperature of the high-density fermentation is 30-37℃, the time of the high-density fermentation is 24-48 h, and the pH of the fermentation system is controlled to be 6.0-6.5 during the high-density fermentation.

4. A method of fermenting a salted pepper juice using the starter culture of claim 1 or the starter culture prepared by the method of claim 2 or 3, characterized in that, The method comprises the following steps: S1) mixing salted pepper juice, fresh sauce, white wine and glucose to obtain a mixture; S2) inoculating the starter into the mixture and sealing for fermentation to obtain a fermentation product; S3) performing solid-liquid separation on the fermentation product to obtain flavor filter sauce and flavor filter liquor respectively.

5. The method of claim 4, wherein, In step S1), the fresh sauce comprises chili sauce and tomato sauce, and the mass ratio of the chili sauce to the tomato sauce is (1-2):(1-2); the mass of the fresh sauce is 60%-100% of the mass of the salted pepper juice. The alcohol content of the white wine is 45%-55%, the mass of the white wine is 1%-4% of the mass of the salted pepper juice, and the mass of the glucose is 2%-6% of the mass of the salted pepper juice.

6. The method of claim 4, wherein, In step S2), the inoculation amount of the starter is 0.1%-2% of the mass of the mixture, and the time of the sealed fermentation is 5-10 d.

7. The flavor filter sauce or the flavor filter liquor obtained by the method of any one of claims 4-6.

8. Use of the flavouring sauce or the flavouring liquor according to claim 7 in the flavouring of chilli products, characterised in that, The flavor filter sauce is used for flavoring and taste-enhancing chili sauce products, and the flavor filter liquor is used for flavoring and taste-enhancing water juice chili products.

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