Lactobacillus plantarum and its use in mixed fermentation of bamboo shoot processing residues

Abstract

The present application relates to a lactobacillus plantarum and its use in mixed fermentation of bamboo shoot processing residues. The lactobacillus plantarum provided by the present application can fully ferment and utilize bamboo shoot processing residues to produce bamboo shoot fermentation liquor, which not only provides raw materials for subsequent production of sour soup base, but also improves the overall utilization rate of bamboo shoots by more than 30%. The sour soup base provided by the present application has a high sensory score, and the color, shape, flavor and taste of the soup base are all excellent, and can produce a unique fragrance flavor, which is different from other sour soup bases on the market, and enriches the types of sour soup base products.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to Lactobacillus plantarum YDSS1 and Lactobacillus plantarum YDSS2 and their use in the preparation of fermented vegetable foods or fermented bamboo shoot processing residues, as well as a sour soup base and its preparation method. Background Technology

[0002] Bamboo shoots, also known as bamboo sprouts, bamboo buds, or bamboo embryos, are the swollen buds and tender stems of plants in the Bambusoideae subfamily of the Poaceae family. They are hailed as the "first-class vegetarian food." Bamboo shoots are rich in dietary fiber, polysaccharides, protein, amino acids, iron, zinc, magnesium, manganese, sterols, polyphenols, flavonoids, and other nutrients and secondary metabolites. They have effects such as regulating intestinal health, reducing cardiovascular disease and diabetes, and possessing antioxidant, anti-inflammatory, and anti-cancer properties. Processed bamboo shoot foods mainly include dried bamboo shoots, canned bamboo shoots, and pickled bamboo shoots. However, currently, to maintain the freshness of the finished product, most bamboo shoots are processed by removing the nodes and sheaths, leaving only the internodes. This results in processing residues accounting for about 50% of the raw material weight, leading to low utilization of biological resources, significant economic losses, and substantial environmental pollution risks. Research shows that the nodes and sheaths have the same nutritional composition as the internodes, and in some cases, the content of certain nutrients is even higher. However, improving the overall utilization rate of bamboo shoots in the food processing industry remains a technical challenge.

[0003] Sour soup bases, as important flavor modifiers in the food industry, possess multiple functional characteristics: First, they activate taste buds to promote saliva secretion, enhancing appetite and flavor profiles; second, they balance the pH value of food to optimize texture (such as tenderizing meat or stabilizing gel structures); third, by creating an acidic environment, they inhibit the proliferation of some spoilage bacteria, extending shelf life. Therefore, they are widely used in hot pot bases, noodle and rice noodle side dishes, and other products. Currently, sour soup bases are mostly based on compound seasonings such as rice vinegar, tomato, and sauerkraut. Soup base products made from bamboo shoot fermentation liquid are not yet widely available. The reason for this is that bamboo shoot fermentation is currently mainly based on natural fermentation, which produces a strong odor (due to p-methylphenol accounting for over 90% of volatile flavor compounds), limiting the consumer base. Furthermore, the natural fermentation process involves a complex variety of microorganisms, and the presence of pathogenic bacteria and viruses in the fermentation system can negatively impact product safety. Summary of the Invention

[0004] The purpose of this invention is to provide a novel *Lactobacillus plantarum* that can ferment bamboo shoot processing residues, thereby improving the utilization rate of bamboo shoot raw materials. At the same time, after fermentation of bamboo shoot processing residues by this *Lactobacillus plantarum*, it can reduce the strong odor and contamination of miscellaneous bacteria present in naturally fermented bamboo shoots. The resulting fermentation product can be used to prepare sour soup bases, possessing a refreshing flavor that is not currently available in commercial products, and can be used to enhance the aroma and / or flavor of food.

[0005] In a first aspect, the present invention provides a *Lactobacillus plantarum*, comprising *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2, wherein *Lactobacillus plantarum* YDSS1 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251282; and *Lactobacillus plantarum* YDSS2 is deposited at the same collection with accession number CCTCC NO: M20251283.

[0006] Of the *Lactobacillus plantarum* species described in this invention, *Lactobacillus plantarum* YDSS1 has the characteristics of producing acid and degrading nitrite, while *Lactobacillus plantarum* YDSS2 has the characteristics of producing acid and generating phenylacetaldehyde and 4-vinylphenol.

[0007] In some embodiments, when the Lactobacillus plantarum YDSS1 ferments bamboo shoot processing residues for 10 days, the pH of the fermentation broth is 3.72 and the total acid content is 8.36 g / L.

[0008] In some embodiments, when the Lactobacillus plantarum YDSS2 ferments bamboo shoot processing residues for 10 days, the pH of the fermentation broth is 3.77 and the total acid content is 8.73 g / L.

[0009] In some embodiments, when the Lactobacillus plantarum YDSS1 ferments bamboo shoot processing residues for 8 hours to 18 days, the nitrite content in the fermentation broth remains at a low level, between 0.34 and 0.43 mg / kg.

[0010] In some embodiments, when the *Lactobacillus plantarum* YDSS2 ferments bamboo shoot processing residues for 10 days, the OAV value of phenylacetaldehyde in the fermentation broth is 8148.50, and the OAV value of 4-vinylphenol is 1655.00.

[0011] In this invention, "bamboo shoot processing residue" refers to the parts discarded in the production of bamboo shoot processed foods, such as bamboo shoot nodes and bamboo shoot sheaths.

[0012] In a second invention, the present invention provides the use of the aforementioned *Lactobacillus plantarum* in (a) or (b):

[0013] (a) Preparing fermented vegetable food, wherein the fermented vegetable food includes fermented bamboo shoot food, sauerkraut, and pickled radish, and the fermented bamboo shoot food includes pickled bamboo shoot, pickled bamboo shoot soup, and sour soup base;

[0014] (b) Fermented bamboo shoot processing residue.

[0015] In some embodiments, the *Lactobacillus plantarum* YDSS1 can increase the total acid content in the fermented vegetable food or the bamboo shoot processing residue and reduce the nitrite content in the fermented vegetable food or the bamboo shoot processing residue.

[0016] In some embodiments, the *Lactobacillus plantarum* YDSS2 can increase the total acid content in the fermented vegetable food or the bamboo shoot processing residue, and increase the content of phenylacetaldehyde and 4-vinylphenol in the fermented vegetable food or the bamboo shoot processing residue.

[0017] In some embodiments, in the described use, the total viable count of the inoculated *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2 is 10. 7 -10 9 The inoculation ratio was (1-2):(1-2), and the total inoculation amount was 1-3% by mass.

[0018] Thirdly, the present invention provides a method for preparing a sour soup base, comprising:

[0019] Provide bamboo shoot processing residues;

[0020] The bamboo shoot processing residue is cleaned, crushed, enzymatically hydrolyzed, and sterilized, then inoculated with Lactobacillus plantarum and fermented to obtain a fermentation broth.

[0021] The fermentation liquid is filtered, and the resulting filtrate is mixed with seasonings, then concentrated, filtered, and sterilized to obtain a sour soup base.

[0022] The cleaning and crushing processes can be carried out according to conventional methods in the art. In some embodiments, the crushing process involves cutting the bamboo shoot processing residue into pieces and juicing it at a material-to-water ratio of 1:2.

[0023] In some embodiments, the enzymatic hydrolysis is performed using a complex enzyme containing pectinase, cellulase, hemicellulase and ligninase in a mass ratio of (1-2):(1-3):(1-3):(1-3), preferably 1:1:1:1.

[0024] In some embodiments, the amount of the complex enzyme added is 1-3% by mass.

[0025] In some embodiments, the enzymatic hydrolysis treatment is carried out at a temperature of 40–45°C for a time of 0.5–1.5 h.

[0026] In some embodiments, the enzymatic hydrolysis process is followed by an enzyme inactivation step, such as boiling in water for 10 minutes.

[0027] In some embodiments, the sterilization process is performed at 121°C for 15 minutes.

[0028] In some embodiments, the inoculation step of *Lactobacillus plantarum* may involve inoculating only *Lactobacillus plantarum* YDSS1 or only *Lactobacillus plantarum* YDSS2 for fermentation; preferably, *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2 are mixed inoculated, and the total viable count of *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2 is 10-1. 7 -10 9 The inoculation ratio was (1-2):(1-2), and the total inoculation amount was 1-3% by mass.

[0029] In some embodiments, the fermentation treatment is carried out at a temperature of 35–39°C, preferably 35°C, for a duration of 7–12 days, preferably 10 days.

[0030] In some embodiments, the seasonings include minced garlic, ginger slices, shallots, Sichuan peppercorns, white sugar, salt, oyster sauce, light soy sauce, and bird's eye chili; preferably, based on the weight of the filtrate, the amount of minced garlic, ginger slices, and shallots added is 1% by mass, and the amount of Sichuan peppercorns added is 0.5% by mass; preferably, the minced garlic, ginger slices, shallots, and Sichuan peppercorns are stir-fried in hot oil; preferably, the amount of white sugar, salt, oyster sauce, light soy sauce, and bird's eye chili added is 0.5-2.5% by mass; more preferably, the amount of white sugar added is 1% by mass, the amount of salt added is 1% by mass, the amount of oyster sauce added is 1% by mass, the amount of light soy sauce added is 0.5% by mass, and the amount of bird's eye chili added is 2% by mass.

[0031] In some embodiments, the concentration filtration and sterilization process is carried out by boiling for 6 minutes.

[0032] In some embodiments, the sterilization process, which involves vacuum packaging followed by sterilization at 121°C for 15 minutes, is described as a process involving concentration, filtration, and sterilization.

[0033] In some embodiments, when the Lactobacillus plantarum YDSS1 is used to ferment the bamboo shoot processing residue separately for 10 days, the pH of the fermentation broth is 3.72 and the total acid content is 8.36 g / L.

[0034] In some embodiments, when the Lactobacillus plantarum YDSS2 is used to ferment the bamboo shoot processing residue separately for 10 days, the pH of the fermentation broth is 3.77 and the total acid content is 8.73 g / L.

[0035] In some embodiments, when the Lactobacillus plantarum YDSS1 is used to ferment the bamboo shoot processing residue separately for 8 hours to 18 days, the nitrite content in the fermentation broth remains at a low level, between 0.34 and 0.43 mg / kg.

[0036] In some embodiments, when the Lactobacillus plantarum YDSS2 is used to ferment the bamboo shoot processing residue separately for 10 days, the OAV value of phenylacetaldehyde in the fermentation broth is 8148.50 and the OAV value of 4-vinylphenol is 1655.00.

[0037] Fourthly, the present invention provides a sour soup base, which is prepared by the preparation method described in the third aspect.

[0038] In some implementations, the sour soup base is used to enhance the flavor and / or season food, including but not limited to hot pot, rice noodles, noodles, and rice vermicelli.

[0039] Compared with existing technologies, the *Lactobacillus plantarum* provided by this invention can fully ferment bamboo shoot processing residues to produce bamboo shoot fermentation liquid, providing raw materials for subsequent production of sour soup bases and increasing the overall utilization rate of bamboo shoots by more than 30%. Among them, *Lactobacillus plantarum* YDSS1 and YDSS2 both have strong acid-producing capabilities, and the total acid accumulation can reach a stable level after 10 days of fermentation of bamboo shoot processing residues. Meanwhile, compared with other Lactobacillus plantarum, Lactobacillus plantarum YDSS1 has a stronger ability to degrade nitrite, which is beneficial to reducing nitrite in fermented foods and improving food safety. Lactobacillus plantarum YDSS2 can produce more volatile flavor substances when fermenting bamboo shoot processing residues, especially phenylacetaldehyde with fruity and rose aromas, and 4-vinylphenol with meaty aroma. This makes the bamboo shoot fermentation liquid have both phenolic and aldehyde aromas while reducing the strong odor produced by natural fermentation. The sour soup base provided by this invention has a high sensory score. The soup base has excellent color, shape, flavor and taste, and can produce a unique fragrance, which is different from other sour soup bases on the market and enriches the variety of sour soup base products.

[0040] Information on strain preservation:

[0041] Lactobacillus plantarum YDSS1 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20251282, deposit date: June 6, 2025.

[0042] Lactobacillus plantarum YDSS2 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20251283, deposit date: June 6, 2025. Attached Figure Description

[0043] Figure 1 Gram-stained microscopic image of Lactobacillus plantarum YDSS1.

[0044] Figure 2 Gram-stained microscopic image of Lactobacillus plantarum YDSS2.

[0045] Figure 3 Phylogenetic tree of Lactobacillus plantarum YDSS1 and Lactobacillus plantarum YDSS2.

[0046] Figure 4 The pH changes in bamboo shoot processing residues fermented with different Lactobacillus strains are shown.

[0047] Figure 5 The study shows the changes in total acidity in bamboo shoot processing residues fermented with different Lactobacillus strains.

[0048] Figure 6 The changes in nitrite levels in bamboo shoot processing residues fermented with different Lactobacillus strains are shown.

[0049] Figures 7A to 7C The total ion chromatogram of volatile flavor compounds in a representative sample of fermented bamboo shoot processing residue is shown. Figure 7A For uninoculated fresh blank samples KB, Figure 7B This is a sample of YDSS1 fermented for 10 days. Figure 7C The sample is from YDSS2 fermentation for 10 days.

[0050] Figure 8 The overall utilization rate of bamboo shoots before and after processing residues is shown.

[0051] Figure 9 The process flow for preparing sour soup base is shown. Detailed Implementation

[0052] The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the invention will become apparent from this specification and the accompanying drawings, and from the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the invention will be practiced using conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and food science, all of which are within the scope of this art.

[0053] Example

[0054] The following embodiments further illustrate the present invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art.

[0055] Unless otherwise specified, the experimental methods described herein are conventional methods in the art using default parameters and procedures; the experimental materials used are commercially available products unless otherwise specified. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art, or according to the corresponding product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0056] Example 1: Isolation, screening, physiological and biochemical identification, and strain characteristics of Lactobacillus plantarum

[0057] 1. Separation and screening

[0058] Naturally fermented pickled bamboo shoot extract from northern Guangdong (purchased from Guanji Bamboo Shoot Factory, Shihuipu Town, Yingde City, Qingyuan City) was used. The extract was aseptically streaked onto MRS solid medium using the S-stripe method and incubated at 37℃ for 72 hours. After colony growth, the morphological characteristics of single colonies were observed, recorded, and photographed promptly. Lactobacillus was preliminarily screened from single colonies using Gram staining and microscopic examination. The colonies were then stored in 40% sterile glycerol at -80℃ for later use.

[0059] 2. Safety evaluation and identification of the strain

[0060] (1) Plasma coagulase test

[0061] The test strains YDSS1 and YDSS2 were cultured separately in MRS liquid medium at 37±1℃ for 24 h, with Staphylococcus aureus as a positive control and sterile MRS liquid medium and physiological saline as negative controls. 0.8 mL of each YDSS1 and YDSS2 bacterial culture was transferred to a vial containing 0.5 mL of lyophilized rabbit plasma, gently shaken, and incubated at 37℃±2℃. Observations were made every 0.5 h for a total of 12 h. Coagulation was considered positive, and no coagulation was considered negative. Results were recorded.

[0062] (2) Hemolysis test

[0063] Under aseptic conditions, the test strain was picked up from the plate and inoculated onto a sterile blood agar plate using an inoculation needle. The plate was then incubated at 37℃±2℃ for 24 hours, and the formation of hemolytic zones was observed. Staphylococcus aureus was used as a positive control. Three parallel experiments were performed for each sample, and the results were recorded.

[0064] (3) Thermoresistant DNAase test

[0065] Make 2 mm diameter wells in toluidine blue-DNA agar medium and remove the agar. Add approximately 0.02 ml of activated broth containing the bacterial culture to be tested to each well, with Staphylococcus aureus as a positive control. Place the plates upright in an incubator and incubate at 37℃±2℃ for 24 h. A pink ring around each well indicates a positive result, otherwise a negative result.

[0066] Table 1

[0067]

[0068] Note: "-" indicates a negative result, and "+" indicates a positive result.

[0069] As shown in Table 1, the safety test results of YDSS1 and YDSS2 isolated and screened from pickled bamboo shoots were both negative, indicating that they are relatively safe strains that can be used for food fermentation.

[0070] The activated strain was inoculated into MRS medium and cultured at 37°C for 24–48 h. ① Gram staining and microscopic examination were performed. ② After freezing and centrifugation, pure bacterial sludge was obtained. DNA was extracted using a bacterial genome extraction kit (Hangzhou Lianchuan Biotechnology Co., Ltd.). The 16S-rDNA sequence of the strain was amplified by PCR using universal primers for bacterial identification 27F (5'-AGTTTGATCMTGGCTCAG-3'(SEQ ID NO:1)) and 1492R (5'-GGTTACCTTGTTACGACTT-3'(SEQ ID NO:2)). 16S-rDNA sequencing was then performed. The sequenced information (the sequence of *Lactobacillus plantarum* YDSS1 is shown in SEQ ID NO:3, and the sequence of *Lactobacillus plantarum* YDSS2 is shown in SEQ ID NO:4) was submitted to the website of the National Center for Biotechnology Information (NCBI). Homology comparison was performed using the basic local alignment search tool (BLAST). Then, the 16S-rDNA gene sequences of known *Lactobacillus* standard strains were downloaded from the GenBank database. A phylogenetic tree was constructed using the Neighborjoining (NJ) bootstrap method with a value of 1000 in MEGA12.0 software to determine the genus-species relationships.

[0071] Gram staining results as follows Figure 1 and 2 As shown, both YDSS1 and YDSS2 showed positive Gram staining results. The bacterial cells of the strains were slender rod-shaped or short and thick rod-shaped, distributed singly, in pairs, or in chains, exhibiting the morphological characteristics of lactobacilli.

[0072] Phylogenetic tree of YDSS1 and YDSS2 as follows Figure 3 As shown, YDSS1 is most closely related to Lactiplantibacillusplantarum 123-17, and YDSS2 is most closely related to Lactiplantibacillusplantarum MSD1-4. They are identified as belonging to the genus Lactobacillus plantarum and are named Lactobacillus plantarum YDSS1 and Lactobacillus plantarum YDSS2, respectively.

[0073] 3. Physiological and biochemical identification

[0074] The physiological and biochemical characteristics of the screened strains were tested in accordance with the "Manual of Systematic Identification of Common Bacteria" and "Classification, Identification and Experimental Methods of Lactic Acid Bacteria". The experiments were carried out in accordance with the experimental steps in the relevant biochemical identification kits. The experiments included the optimal temperature, optimal pH, salt tolerance test of 5% and 10% NaCl, catalase test, gelatin liquefaction test, glucose gas production test, nitrate reduction test, and indole test.

[0075] Optimal temperature: Following the methods of Xiang Huanling, Yu Peibin, and Liu Shaojie, the dominant bacteria were inoculated into liquid culture medium at an inoculum rate of 3% (v / v) and cultured at 27, 30, 35, 37, 42, and 47℃ for 48 h, respectively, with uninoculated liquid culture medium used as zeroing. The OD value of the culture medium was measured at a wavelength of 600 nm. A curve was plotted with OD600 nm value on the ordinate and temperature on the abscissa.

[0076] Optimal pH: The pH of the liquid culture medium was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 using 1 mol / L HCl and 1 mol / L NaOH solutions, respectively. After two generations of activation by inoculating the dominant bacterial strain, the culture medium was inoculated at a rate of 3% (v / v) into each gradient of liquid culture medium and cultured at 37°C for 48 hours. The growth of the strain was observed. The OD value of the bacterial solution was measured at 600 nm using a spectrophotometer. A curve was plotted with OD600 nm as the ordinate and pH value as the abscissa.

[0077] Salt tolerance tests at 5% and 10% concentrations: Activated third-generation bacterial cultures were inoculated at 3% (v / v) into liquid culture media containing 5% and 10% NaCl, respectively. Lactobacillus was cultured on MRS medium, and Staphylococcus on NB medium. Incubation was performed at 37°C for 48 hours. The presence of colonies was considered positive, while the absence of colonies was considered negative. The OD value of the bacterial cultures was measured using a spectrophotometer at 600 nm.

[0078] Catalase test: Using an inoculation loop, scrape a portion of the activated bacterial culture onto a slide pre-treated with 3% H2O2. Mix thoroughly with a sterile toothpick and observe immediately. The appearance of bubbles within 30 seconds indicates a positive reaction; otherwise, it is negative. Gelatin liquefaction test: Scrape fresh bacterial culture with an inoculation needle and inoculate into an identification tube. Incubate at 37°C for 48–72 hours, then place in a 4°C refrigerator for 10 minutes and observe the results. Liquefaction indicates a positive result; coagulation indicates a negative result. A blank control group is also set up, containing sterile physiological saline.

[0079] Glucose gas production test: Before use, ensure that the small inverted tube in the identification tube is free of air bubbles. After activating the strain, inoculate it into the identification tube according to the instructions, incubate at 37°C for 24 hours, and observe the color change in the tube and whether there are air bubbles in the small inverted tube. A blank control tube should also be set up. The presence of air bubbles in the small inverted tube indicates a positive reaction, while the absence of air bubbles indicates a negative reaction.

[0080] Nitrate reduction experiment: After the strain is activated, it is inoculated into a biochemical identification tube. The biochemical tube is partially stoppered and incubated at 37°C for 18-24 hours. Then, 2 drops each of nitrate reduction reagent A and B are added, mixed well, and the results are observed immediately.

[0081] Indole test: Inoculate approximately 0.08 mL of activated bacterial solution into a peptone-water biochemical identification tube. Incubate at 37°C for 24 hours. Add 2-3 drops of indole reagent to the tube, gently shake the vial, and observe the color change. Use sterile physiological saline as a blank control. A red color indicates a positive result; no color change indicates a negative result.

[0082] Results: YDSS1 and YDSS2 exhibited optimal growth temperatures between 35 and 37°C, with growth gradually decreasing at temperatures ≥42°C. Both YDSS1 and YDSS2 grew within a pH range of 4–9, with optimal growth at pH 5, after which the OD value gradually decreased. Table 2 shows that YDSS1 and YDSS2 produced no bubbles in the catalase test; both showed colony growth in the 5% NaCl test, indicating some tolerance to NaCl; only slight growth was observed in 10% NaCl, with YDSS2 showing almost no colony growth; neither YDSS1 nor YDSS2 caused gelatin liquefaction; and both YDSS1 and YDSS2 were negative in the nitrate reduction test.

[0083] Table 2

[0084]

[0085] Note: "+" indicates a positive result or growth; "-" indicates 90% negative or no growth; "±" indicates a weakly positive result or slight growth.

[0086] 4. Characteristics of the strain

[0087] The characteristics of the strains were determined by fermenting bamboo shoot processing residues with different Lactobacillus strains. The bamboo shoot processing residues were treated as follows:

[0088] After cleaning the remaining bamboo shoot sections and outer sheaths, cut them into appropriately sized pieces. Juice the bamboo shoots at a weight ratio of 1:2 (material to water). Add 1-3% of a compound enzyme (pectinase, cellulase, hemicellulase, and ligninase in a 1:1:1:1 ratio). Enzymatically hydrolyze the mixture at 60℃ for 0.5-1.5 hours, then boil in water for 10 minutes to inactivate the enzymes, obtaining the enzymatic hydrolysate. Sterilize the hydrolysate at 121℃ for 15 minutes, cool to room temperature, and inoculate with Lactobacillus (single-strain inoculation, 10...). 7 ~10 9 The inoculum was 1-3% by mass (cfu / mL) and fermented at a constant temperature of 35°C.

[0089] (1) Acid production

[0090] pH: Measured using a pH meter; Total acid: Determined using acid-base indicator titration method according to GB 12456-2021 "National Food Safety Standard - Determination of Total Acid in Food".

[0091] The pH variation of different lactobacillus strains (YDSS1 and YDSS2 are Lactobacillus plantarum, isolated and screened from pickled bamboo shoots in this study; the other 7 lactobacillus strains are Levilactobacillus spicheri, isolated and screened from pickled bamboo shoots in previous studies, and reported in Food Chemistry, 2024, 437:137858) in the fermentation residue of bamboo shoots was determined (see...). Figure 4 As the fermentation process progressed, the pH value of the bamboo shoot fermentation residue from different lactobacilli gradually decreased, reaching a stable low level (pH value between 3.72 and 3.89) after 10 days of fermentation. YDSS2 and YDSS1 had even lower pH values ​​than the other samples, at 3.77 and 3.72, respectively. Conversely, throughout the fermentation process, the total acid content of the bamboo shoot processing residue from different lactobacilli gradually increased (see...). Figure 5 By fermentation day 10, the total acid content reached a stable and relatively high level (between 6.20 and 8.73 g / L). Among them, the total acid content of YDSS2 and YDSS1 was relatively high, reaching 8.73 g / L and 8.36 g / L respectively. This corresponds to the pH analysis results, indicating that different lactobacilli have different acid production capabilities under the same fermentation conditions. YDSS2 and YDSS1 strains have strong acid production capabilities, and their total acid accumulation reached a stable level after 10 days of fermentation.

[0092] (2) Nitrite

[0093] Nitrite content: Refer to the national standard GB 5009.33-2016 "Determination of Nitrite and Nitrate in Food" and use spectrophotometry.

[0094] Nitrites are harmful substances produced during vegetable fermentation; therefore, fermented products should ensure that the nitrite content is within safe limits. Figure 6 As shown, throughout the fermentation process, the nitrite content in the bamboo shoot processing residues fermented by the nine different lactobacilli ranged from 0.34 to 0.97 mg / kg, all within the nationally stipulated safety level (≤20 mg / kg). Among them, YDSS1 had the lowest nitrite content (0.34 mg / kg), indicating that it has the ability to degrade nitrite.

[0095] (3) Volatile flavor compounds

[0096] Sample pretreatment: Liquid-liquid extraction (LLE) was used. 2 mL of fermentation broth was placed in a 10 mL volumetric flask, and 5 mL of a mixed extractant (chromatographically pure ethanol and dichloromethane in a 1:1 ratio) was added simultaneously. The mixture was sonicated at 30°C for 10 minutes, and then brought to a final volume of 10 mL with the mixed extractant. The diluted extract was thoroughly mixed and filtered through a 22 mm diameter, 0.22 μm pore size organic filter membrane into a 1.5 mL injection vial. The vial was sealed and stored at 4°C. 1 μL of the sample was injected directly.

[0097] GC conditions: Column: DB-WAX (30m×0.25mm×0.25μm); Carrier gas: He, purity 99.999%; Carrier gas flow rate: 1mL / min, constant flow 36.877cm / sec; Injector temperature: 230℃; Injection mode: split, split ratio 10:1; Temperature program: initial temperature 50℃, hold for 1 min, increase to 230℃ at 10℃ / min, hold for 3 min; Injection volume: 1.0μm.

[0098] MS conditions: Interface temperature, 250℃; Scan mode, SIM; Acquisition quality range, 5~350amu; Ion source, electron impact ionization (EI), ionization energy, 70eV; Ion source temperature, 230℃; Solvent delay 4min.

[0099] Qualitative analysis: The total ion chromatogram was used to identify the components retrieved from the fermentation broth using the NIST20 standard spectral library. The compounds were determined by combining the retention index (RI), which was calculated according to formula (1).

[0100]

[0101] t i Adjust the retention time for the analyte compound; t n Adjust the retention time for n-carbon alkanes; t n+1 Adjust the retention time for n+1 carbon atoms in n-alkanes; n is the number of carbon atoms.

[0102] Quantitative analysis: Quantitative analysis was performed using a combination of external standard and internal standard methods. A mixture of volatile flavor compounds (containing acetic acid) with an OAV ≥ 1 and a concentration of 0.1 mg / kg to 10 g / kg was analyzed under the GC and MS conditions described above. A standard curve of volatile flavor compound concentration versus peak area was established, and the external standard was used to determine the concentration. For the other volatile flavor compounds, the acetic acid content accurately measured by the external standard method was used as the internal standard reference, and the concentration was calculated using formula (2), where C... i C represents the concentration of a substance in the sample being measured, in μg / g. 乙酸 A represents the concentration of acetic acid in the sample being measured, in μg / g; i A is the peak area of ​​the target substance; A0 is the peak area of ​​acetic acid.

[0103]

[0104] Characteristic flavor compounds are determined by the OAV value, and the calculation formula is shown in (3), where: C i The concentration of this volatile flavor compound is expressed in μg / g; OT i OAV represents the flavor threshold of the volatile flavor compound, expressed in μg / g. When OAV > 1, it indicates that the compound is a characteristic flavor compound that contributes significantly to the overall flavor of the sample; when 0.1 ≤ OAV < 1, it indicates that the compound contributes to the flavor of the pickled bamboo shoot soup sample to a certain extent; when OAV < 0.1, it indicates that the compound has little or no effect on the overall flavor of the sample.

[0105]

[0106] Volatile flavor compounds were detected in bamboo shoot fermentation residues from different Lactobacillus strains after 10 days. The total ion chromatogram is shown below. Figures 7A-7C Therefore, the good peak shapes of each substance indicate good separation, and the selected method is suitable for the determination of volatile flavor compounds in fermented bamboo shoot pulp. The substances with OAV ≥ 1 in bamboo shoot pulp fermented by different lactobacilli for 10 days were summarized (see Tables 3 and 4). A total of 13 substances in the bamboo shoot pulp fermented by 9 lactobacilli showed OAV ≥ 1 in at least one sample. Among them, 6 substances in YDSS2 showed OAV ≥ 1, especially phenylacetaldehyde and 4-vinylphenol, whose OAV values ​​were as high as 8148.50 and 1655.00 respectively, at least 1.5 times and 2.4 times higher than those in other lactobacilli fermented bamboo shoot pulps. Phenylacetaldehyde mainly has fruity and rose-like aromas, while 4-vinylphenol has a meaty aroma. These two substances greatly enhance the phenolic and aldehyde aromas of YDSS2 fermented bamboo shoot pulp.

[0107] Table 3. Content of main flavor compounds in bamboo shoot processing residues fermented with different Lactobacillus strains (10 days)

[0108]

[0109]

[0110] Note: - indicates that the substance was not detected; N indicates that no information about the compound was found; RIA is calculated according to formula (1); RIB is obtained from https: / / www.nist.gov / .

[0111] Table 4. Description of main flavor compounds, odor threshold, and OAV value of bamboo shoot processing residues fermented with different Lactobacillus inoculations (10 days).

[0112]

[0113]

[0114] Note: - indicates that the substance was not detected; N indicates that no information related to the compound was found; the threshold data mainly comes from the "Compilation of Compound Olive Thresholds", and the OAV value is calculated according to formula (3).

[0115] Example 2: Preparation of Sour Soup Base

[0116] 1. Experimental Methods

[0117] 1.1 Preparation and enzymatic hydrolysis of bamboo shoot pulp

[0118] After cleaning the remaining bamboo shoot sections and sheaths from bamboo shoot processing, cut them into appropriately sized pieces. Juice them at a material-to-water ratio of 1:2, add 1-3% of a compound enzyme (pectinase, cellulase, hemicellulase, and ligninase in a 1:1:1:1 ratio), and enzymatically hydrolyze at 60℃ for 0.5-1.5 hours. Then boil in water for 10 minutes to inactivate the enzymes, and obtain bamboo shoot slurry enzymatic hydrolysate for later use.

[0119] 1.2 Fermentation

[0120] The bamboo shoot pulp enzymatic hydrolysate was sterilized at 121℃ for 15 minutes, cooled to room temperature, and then inoculated with *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2 (total viable count of 10). 7 -10 9 The bamboo shoot fermentation liquid (cfu / mL, ratio 1:1, addition amount 2% by mass) was fermented at a constant temperature of 35℃. The fermentation liquids of bamboo shoots were collected after fermentation for 8h, 16h, 1d, 1d+8h, 1d+16h, 2d, 2d+8h, 2d+16h, 3d, 5d, 7d, 10d, 12d, 15d, and 18d, and their physicochemical and flavor indicators were measured to screen the optimal fermentation time.

[0121] 1.3 Determination of the overall utilization rate of bamboo shoots after processing residue

[0122] The overall utilization rate of bamboo shoots after processing residue is calculated using formula (4), where x represents the overall utilization rate of bamboo shoots, %; RM T This indicates the total raw material quantity of bamboo shoots, expressed in kg; RM S This refers to the filter residue after filtering the fermentation liquid from bamboo shoot processing, expressed in kg.

[0123]

[0124] The overall utilization rate of bamboo shoots before and after processing residues is as follows: Figure 8 As shown, it can be seen that by using discarded bamboo shoot segments, bamboo shoot sheaths, and other bamboo shoot processing residues to produce bamboo shoot fermentation liquid, the overall utilization rate of bamboo shoots can be increased by more than 30%.

[0125] 2. Formula and optimization of sour soup base packets

[0126] The bamboo shoot fermentation liquid obtained in step 1.2 was filtered. The filtrate was then added to a mixture of garlic (1%), ginger slices (1%), shallots (1%), and Sichuan peppercorns (0.5%) that had been sautéed in hot oil until fragrant. The proportions of white sugar, salt, oyster sauce, light soy sauce, and chili peppers were optimized. The mixture was then boiled and concentrated for 6 minutes. The residue was filtered off, and the mixture was vacuum-packed and sterilized at 121°C for 15 minutes to obtain the sour soup base. The preparation process of the sour soup base is as follows: Figure 9 As shown.

[0127] 2.1 Formula Optimization

[0128] Based on the preparation process of the sour soup base, a stepwise optimization method was adopted using individual factors, with sensory evaluation as the indicator, to optimize five formula factors: white sugar addition (0.5%, 1.0%, 1.5%, 2.0%, 2.5%), salt addition (0.5%, 1.0%, 1.5%, 2.0%, 2.5%), oyster sauce addition (0.5%, 1.0%, 1.5%, 2.0%, 2.5%), light soy sauce addition (0.25%, 0.50%, 0.75%, 1.00%, 1.25%), and millet pepper addition (0.5%, 1.0%, 1.5%, 2.0%, 2.5%).

[0129] 2.2 Sensory Evaluation Methods

[0130] The prepared sour soup base was diluted with water at a ratio of 1:5. After boiling, the rice noodles were cooked for 8 minutes. Fifteen professionals were selected to taste and evaluate the food using the evaluation criteria in Table 5. Each evaluator should clean their mouth immediately after completing the evaluation of a single sample before continuing to evaluate subsequent samples.

[0131] Table 5 Sensory Evaluation

[0132]

[0133] 2.3 Experimental Results

[0134] 2.3.1 Effect of white sugar addition on the sensory quality of sour soup base

[0135] Table 6 shows the effect of different amounts of added white sugar on the sensory quality of sour soup base when the ingredients are 1% salt, 1.5% millet pepper, 1.5% oyster sauce, and 0.75% light soy sauce. When the amount of added white sugar is 1%, the sensory score of the sour soup base is higher, and the color, appearance, flavor, and taste of the soup base are better. Therefore, 1% white sugar is selected as the optimal amount.

[0136] Table 6. Effect of white sugar on sensory scores of sour soup base

[0137]

[0138] 2.3.2 Effect of Salt Addition on Sensory Quality of Sour Soup Base

[0139] Table 7 shows the effect of different salt addition amounts on the sensory quality of the sour soup base when the salt content is 1% white sugar, 1.5% millet pepper, 1.5% oyster sauce, and 0.75% light soy sauce. When the salt addition amount is 1%, the overall sensory score of the sour soup base is relatively high, and the rice noodles cooked at this level have a suitable sweet and salty flavor. Therefore, 1% salt addition is selected as the optimal amount.

[0140] Table 7. Effects of salt on sensory scores of sour soup bases

[0141]

[0142] 2.3.3 Effect of the amount of millet pepper added on the sensory quality of sour soup base

[0143] Table 8 shows the effect of different amounts of millet pepper added on the sensory quality of the sour soup base when the ingredients are 1% white sugar, 1% salt, 1.5% oyster sauce, and 0.75% light soy sauce. When the amount of millet pepper added is 2%, the sour soup base has a suitable spiciness and a high sensory score. Furthermore, when the amount of millet pepper added is 0.5–1.5%, although the spiciness is lower and the sensory score is also lower, it is more suitable for people who do not eat or eat little spicy food.

[0144] Table 8. Effect of millet pepper on sensory scores of pickled bamboo shoot soup seasoning packets

[0145]

[0146] 2.3.4 Effect of Oyster Sauce Addition Amount on Sensory Quality of Sour Soup Base

[0147] Table 9 shows the effect of different amounts of oyster sauce added on the sensory quality of the sour soup base packet, with 1% white sugar, 1% salt, 2% millet peppers, and 0.75% light soy sauce. The sensory score of the sour soup base reached its highest when the amount of oyster sauce added was 1%. Under this condition, the color, appearance, flavor, and texture of the soup base were optimal. Increasing the amount added resulted in a too-dark color and excessive saltiness in the sour soup, while too little added resulted in an unbalanced flavor. Therefore, 1% oyster sauce was selected as the optimal addition amount.

[0148] Table 9. Effects of oyster sauce on sensory scores of sour soup base packets.

[0149]

[0150] 2.3.5 Effect of soy sauce addition on the sensory quality of sour soup base

[0151] Table 10 shows the effect of different amounts of light soy sauce added on the sensory quality of the sour soup base when the ingredients are 1% white sugar, 1% salt, 2% millet pepper, and 1% oyster sauce. When the amount of light soy sauce added reaches 0.5%, the sensory score of the sour soup base is relatively high.

[0152] Table 10. Effect of light soy sauce on sensory scores of sour soup bases

[0153]

[0154] Based on the above research, the optimal formula for sour soup base is: 1% minced garlic, 1% ginger slices, 1% shallots, 0.5% Sichuan peppercorns, 1% white sugar, 1% salt, 2% bird's eye chili, 1% oyster sauce, and 0.5% light soy sauce.

Claims

1. A composition of *Lactobacillus plantarum*, comprising *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2, wherein, The Latin name of Lactobacillus plantarum YDSS1 is Lactobacillus plantarum YDSS1 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251282; the Latin name of *Lactobacillus plantarum* YDSS2 is... Lactobacillus plantarum YDSS2, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M 20251283; the composition is used for fermenting bamboo shoots.

2. Use of the composition of claim 1 in the preparation of fermented bamboo shoot food or fermented bamboo shoot processing residues.

3. The use according to claim 2, wherein, In the composition, the total viable count of *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2 is 10. 7 -10 9 The inoculation ratio was (1~2):(1~2), and the total inoculation amount was 1~3% by mass.

4. A method for preparing a sour soup base, comprising: Provide bamboo shoot processing residues; The bamboo shoot processing residue is cleaned, crushed, enzymatically hydrolyzed, and sterilized, then inoculated with the composition of claim 1 and fermented to obtain a fermentation liquid; The fermentation liquid is filtered, and the resulting filtrate is mixed with seasonings, then concentrated, filtered, and sterilized to obtain a sour soup base.

5. The preparation method according to claim 4, wherein, The enzymatic hydrolysis is performed using a complex enzyme containing pectinase, cellulase, hemicellulase, and ligninase in a mass ratio of (1~2):(1~3):(1~3):(1~3), with the amount of the complex enzyme added being 1~3% by mass; the enzymatic hydrolysis is performed at a temperature of 40~45℃ for 0.5~1.5 h.

6. The preparation method according to claim 4, wherein, During the inoculation, the total viable count of *Lactobacillus plantarum* YDSS1 and *Lactobacillus plantarum* YDSS2 was 10. 7 -10 9 cfu / mL, inoculation ratio of (1~2):(1~2), total inoculation amount of 1~3% by mass% The fermentation process is carried out at a temperature of 35-39°C for 7-12 days.

7. The preparation method according to claim 4, wherein, The seasonings include minced garlic, ginger slices, shallots, Sichuan peppercorns, white sugar, salt, oyster sauce, light soy sauce, and chopped chili peppers.

8. The preparation method according to claim 7, wherein, Based on the weight of the filtrate, the amount of minced garlic, ginger slices and shallots added is 1% by mass, and the amount of Sichuan peppercorns added is 0.5% by mass.

9. The preparation method according to claim 7, wherein, The minced garlic, ginger slices, shallots, and Sichuan peppercorns are stir-fried in hot oil.

10. The preparation method according to claim 7, wherein, The amounts of white sugar, salt, oyster sauce, light soy sauce, and chili peppers added are 0.5 to 2.5% by weight, respectively.

11. The preparation method according to claim 10, wherein, The amount of white sugar added is 1% by mass, the amount of salt added is 1% by mass, the amount of oyster sauce added is 1% by mass, the amount of light soy sauce added is 0.5% by mass, and the amount of millet pepper added is 2% by mass.

12. A sour soup base, prepared by the preparation method according to any one of claims 4-11.

13. The sour soup base according to claim 12, wherein, The sour soup base is used to enhance the aroma and / or flavor of food, including hot pot, rice noodles, noodles, and rice vermicelli.

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