Bacillus subtilis deserti subsp. E1-8, fermentation broth, enzymatic hydrolysis product of fermentation broth, preparation method and application thereof

Abstract

The application discloses a bacillus subtilis var. desicus E1-8, a bacillus subtilis var. desicus fermentation liquor, a bacillus subtilis var. desicus fermentation liquor enzymolysis product, a preparation method of the bacillus subtilis var. desicus fermentation liquor enzymolysis product and application of the bacillus subtilis var. desicus fermentation liquor enzymolysis product in fresh-cut fruit preservation. The bacillus subtilis var. desicus E1-8 has a preservation number of CCTCC NO: M20232071. A fermentation method for the bacillus subtilis var. desicus is provided, and the fermentation medium comprises the following components: a carbon source 0.5-5%, a nitrogen source 0.5-5%, a pH value 6-8, a fermentation temperature 28-37 DEG C and 180 r / min oscillation culture. The bacillus subtilis var. desicus strain E8-1 with high yield of extracellular protease is first isolated and identified; the extracellular protease generated by the fermentation of the E8-1 is identified as serine protease and metalloprotease; a fermentation method and parameters for the fermentation of the bacillus subtilis var. desicus strain E8-1 with high yield of extracelluar protease are proposed and optimized; a preparation method of the E8-1 extracellular protease enzymolysis product is proposed and optimized; and the application of the prepared enzymolysis product in fruit preservation is explored.

Description

Technical Field

[0001] This invention relates to the fields of microbial and fruit preservation technology, and in particular to a strain of Bacillus subtilis desert subsp., Bacillus subtilis desert subsp. fermentation broth, enzymatic hydrolysis products of Bacillus subtilis desert subsp. fermentation broth, their preparation methods, and their application in the preservation of fresh-cut fruits. Background Technology

[0002] Freshly cut fruit is convenient and quick to consume, satisfying consumers' nutritional needs in a short time. With the fast pace of life, freshly cut fruit is gaining increasing attention in China, especially among younger consumers. However, freshly cut fruit is typically sold on refrigerated shelves. During this process, surface oxidation and low-temperature damage reduce its appearance and freshness, resulting in an overall quality inferior to whole fruit.

[0003] Currently, most commonly used preservatives for fresh-cut fruits are chemical reagents, such as sulfites, citric acid, and ascorbic acid. Commonly used low-temperature preservatives include polyphosphates, sugars, alcohols, and their compounds. Using chemical additives to treat fresh-cut fruits not only affects their taste, but their residues may also impact human health. Ingestion of sulfites and polyphosphates can worsen symptoms in patients with hypertension and chronic kidney disease; sugar alcohols make products overly sweet and high in energy; and high concentrations of sugar alcohols and polyphosphate antifreeze agents can damage the original structure of food, causing nutrient loss and a decline in the quality of refrigerated foods. Therefore, the development of healthier preservatives for fresh-cut fruits is imperative.

[0004] Plant-derived peptides have attracted widespread attention from researchers both domestically and internationally due to their natural, healthy, and readily available characteristics. The functions of peptides are becoming increasingly clear. Soy peptides possess antibacterial, antihypertensive, lipopolysaccharide-neutralizing, and angiogenesis-promoting physiological activities; pea peptides possess antibacterial, antioxidant, anti-fatigue, and muscle-enhancing activities. Animal-derived peptides can be used as food supplements and functional food ingredients. Fish skin peptides exhibit strong DPPH scavenging, DPP-IV inhibitory, ACE inhibitory, and antioxidant activities, making them suitable as functional food bioactive peptides, antifreeze agents, and antioxidants. They also show potential applications in weight and hyperglycemia management. Porcine skin peptides possess good antifreeze properties, antioxidant, anti-aging, skin penetration characteristics, and ACE inhibitory capabilities, making them suitable as functional peptides for treating skin aging. Research on Bacillus subtilis (Bacillus inaquosorum) mainly focuses on its biocontrol and plant growth-promoting functions. No research has been reported on the proteases produced by Bacillus subtilis and their applications.

[0005] Therefore, if Bacillus subtilis desert subsp., Bacillus subtilis desert subsp. fermentation broth (mainly composed of extracellular proteases), and fermentation broth enzymatic hydrolysis products (plant-derived / animal-derived polypeptides) are developed into preservatives for fresh-cut fruits, they have broad application prospects in the fresh-cut fruit processing industry. Moreover, compared with traditional chemical additives, fermentation broth enzymatic hydrolysis products (plant-derived / animal-derived polypeptides) can also improve the nutritional and economic value of fresh-cut fruits, making them worthy of promotion and application. Summary of the Invention

[0006] This invention aims to solve the aforementioned problems. We screened a high-protease-producing Bacillus subsp. deserte E1-8 strain from the coastal mudflats of Jiaozhou Bay. Through genome sequencing and proteomic analysis, we found that the extracellular proteases in its fermentation broth were serine proteases and metalloproteinases. We improved protease yield by optimizing the fermentation conditions of the strain, and further improved the enzymatic hydrolysis efficiency of animal and plant proteins by optimizing the enzymatic hydrolysis conditions. We established preparation processes for enzymatic hydrolysates of soybeans, peas, fish skin, and pig skin, and explored the application of the prepared enzymatic hydrolysates in the preservation of fresh-cut fruits.

[0007] This invention specifically provides a strain of Bacillus subtilis desert subsp. E1-8, a fermentation broth of Bacillus subtilis desert subsp. E1-8, an enzymatic hydrolysis product of the fermentation broth of Bacillus subtilis desert subsp. E1-8, their preparation methods, and their application in the preservation of fresh-cut fruits.

[0008] In a first aspect, this invention provides a strain of bacteria that produces a high amount of protease, screened from the coastal mudflats of Jiaozhou Bay. This strain was identified as *Bacillus subtilis* desert subspecies and named *Bacillus subtilis* desert subspecies E1-8, with accession number CCTCC NO:M 20232071, hereinafter referred to as E1-8. This bacterium was deposited on October 30, 2023, at the China Center for Type Culture Collection (CCTCC), address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. The requesting institution for deposit was Qingdao Agricultural University.

[0009] Secondly, the present invention provides a fermentation method for Bacillus subtilis desert subspecies E1-8 as described above, wherein the fermentation medium comprises the following components: 0.5% to 5% carbon source, 0.5% to 5% nitrogen source; pH 6 to 8; fermentation temperature 28℃ to 37℃; and shaking culture at 180 r / min.

[0010] Furthermore, the carbon source of the fermentation medium is any one of sweet potato, rice bran, mung bean, wheat bran, yeast powder, xanthan gum, sucrose, and sorbitol; the nitrogen source is any one of soybean meal, peanut meal, fish meal, casein, gelatin, soy protein, pea protein, peptone, and skim milk. Most preferably, the fermentation medium comprises the following components: 3.5% sweet potato as the carbon source, 0.5% soybean meal as the nitrogen source, a fermentation temperature of 37°C, and a pH of 7.0; cultured with shaking at 180 rpm. Compared to the initial medium, the optimized medium reduces costs. The protease activity of the above-mentioned optimal fermentation medium under optimal fermentation conditions can reach up to 321.48 U / ml.

[0011] Thirdly, the present invention also provides a fermentation broth of Bacillus subtilis desert subspecies E1-8, which is prepared from Bacillus subtilis desert subspecies E1-8 as described in the first aspect and the fermentation method described in the second aspect.

[0012] Furthermore, the fermentation broth mainly contains two extracellular proteases: serine proteases and metalloproteases.

[0013] Fourthly, the present invention also provides an enzymatic hydrolysis product of Bacillus subtilis desert subsp. E1-8 fermentation broth, obtained by enzymatic hydrolysis of plant-derived and / or animal-derived protein substrates from the Bacillus subtilis desert subsp. E1-8 fermentation broth described in the third aspect.

[0014] Furthermore, the preparation method of the enzymatic hydrolysis product of Bacillus subtilis desert subsp. E1-8 fermentation broth includes the following steps:

[0015] S1: Prepare plant- and / or animal-derived protein substrates, chop and pre-treat them;

[0016] S2: Add the Bacillus subtilis desert subsp. E1-8 fermentation broth as described in claim 4 for hydrolysis, and set the hydrolysis parameters;

[0017] S3: Thermal deactivation at 90℃ for 15 min;

[0018] S4: Centrifuge at 12000 rpm for 30 min to obtain the supernatant;

[0019] S5: Vacuum freeze-drying yields the enzymatic hydrolysis product of Bacillus subsp. desertis E1-8 fermentation broth.

[0020] The enzymatic hydrolysis product of the Bacillus subtilis desert subspecies E1-8 fermentation broth is preferably a protein peptide freeze-dried powder.

[0021] Further optimization of parameters: In step S3, the hydrolysis parameters for plant-derived products are: pH 11.0–12.0, 60℃, 50–60 min, fermentation broth 800 U / g; the hydrolysis parameters for animal-derived products are: pH 9.0–11.0, 50℃–60℃, 50 min, fermentation broth 600–1000 U / g.

[0022] Preferred optimal parameters: Hydrolysis parameters in step S3:

[0023] Plant-based soy protein: pH 12.0, 60℃, 60 min, 800 U / g (fermentation broth / soy protein); Plant-based pea protein: pH 11.0, 60℃, 50 min, 800 U / g (fermentation broth / pea protein). Animal-based fish skin: pH 9.0, 50℃, 50 min, 1000 U / g (fermentation broth / fish skin); Animal-based pig skin: pH 11.0, 60℃, 50 min, 600 U / g (fermentation broth / pig skin).

[0024] Fifthly, as described in the fourth aspect, the application of the enzymatic hydrolysis product of Bacillus subsp. desertis E1-8 fermentation broth in the preservation of fresh-cut fruits.

[0025] Furthermore, the fresh-cut fruit preservation application includes preventing browning of fresh-cut fruit.

[0026] More preferably, the fresh-cut fruit includes apples and bananas.

[0027] The present invention has the following beneficial effects:

[0028] 1. This invention is the first to isolate and identify Bacillus subtilis desert subspecies E8-1, which produces a high amount of extracellular protease, and optimizes the fermentation method and parameters of Bacillus subtilis desert subspecies E8-1; under the above-mentioned optimal fermentation conditions, the protease activity can reach up to 321.48 U / ml;

[0029] 2. The extracellular protease produced by fermentation of Bacillus subtilis desert subspecies E8-1 of the present invention was identified as a serine protease and a metalloproteinase;

[0030] 3. This invention innovatively proposes and optimizes a method for preparing the enzymatic hydrolysis products of E8-1 extracellular protease;

[0031] 4. This invention is the first to propose the application of E8-1 extracellular protease hydrolysis products in the preservation of fresh-cut fruits. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0033] Figure 1 Example 2 of the present invention: 16S rDNA sequence homology alignment and hydrolysis ability.

[0034] Figure 2 Example 3 of this invention optimizes the fermentation conditions for the production of extracellular protease by Bacillus subsp. deserte E1-8; wherein, (A) the effect of different carbon sources on enzyme production by strain E1-8; (B) the effect of different nitrogen sources on enzyme production by strain E1-8; (C) the effect of sweet potato content and soybean meal content on enzyme production by strain E1-8; and (D) the effect of pH and temperature on enzyme production by strain E1-8.

[0035] Figure 3 Example 3 of this invention: Orthogonal experimental screening of fermentation medium; wherein, (A) protease activity in fermentation broth supernatant; (B) changes in protease activity and secretion results under optimal fermentation conditions.

[0036] Figure 4 Flowchart of the preparation method of enzymatic hydrolysis product of Bacillus subtilis fermentation broth in Example 5 of the present invention.

[0037] Figure 5 Example 5 of this invention optimizes the parameters of the preparation method of enzymatic hydrolysis product of Bacillus subsp. deserte E1-8 fermentation broth; wherein, (A) reaction temperature; (B) pH; (C) enzyme / substrate ratio; (D) reaction time, and the graph shows the data (mean ± standard deviation) of three experiments.

[0038] Figure 6 The molecular weight distribution of four enzymatic hydrolysates of the Bacillus subtilis desert subsp. E1-8 fermentation broth in Example 6 of this invention is shown in the figure. (A) Soybean; (B) Pea; (C) Fish skin; (D) Pig skin.

[0039] Figure 7 The antioxidant activity of the fermentation broth and the enzymatic hydrolysis product of the fermentation broth in Example 7 of this invention; wherein (A) soybean; (B) pea; (C) fish skin; (D) pig skin; (E) fermentation broth.

[0040] Figure 8 Example 8 of this invention: Detection and analysis of the antifreeze ability of the fermentation broth and the enzymatic hydrolysis product of the fermentation broth; wherein (A) soybean; (B) pea; (C) fish skin; (D) pig skin; (E) fermentation broth.

[0041] Figure 9 The freezing point changes of the fermentation broth and the enzymatic hydrolysis products of the fermentation broth in Example 8 of this invention; wherein (A) soybean; (B) pea; (C) fish skin; (D) pig skin; (E) fermentation broth.

[0042] Figure 10 Example 9 of this invention describes the application of fermentation broth and fermentation broth enzymatic hydrolysis products in preventing oxidative browning of fresh-cut fruits; wherein, (A) the effect immediately after spraying; (B) the effect after 24 hours of spraying; (C) the effect after 48 hours of spraying; and (D) the effect at different time points after spraying the fermentation broth.

[0043] Figure 11 Example 9 of the present invention describes the application of the fermentation broth enzymatic hydrolysis product in reducing the browning index of fresh-cut apples; wherein (A) the storage temperature is 4°C; and (B) the storage temperature is 25°C.

[0044] Figure 12-13 The fermentation broth enzymatic hydrolysis product of Example 9 of this invention has a low-temperature preservation effect on banana slices. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0046] Example 1: Isolation and screening of Bacillus inaquosorum E1-8

[0047] Screening process: Soil samples were collected from the coastal mudflats of Jiaozhou Bay, Qingdao City, Shandong Province, at a depth of 2-20 cm. Under aseptic conditions, 10.0 g of sample was weighed and serially diluted in 90 ml of sterile seawater. Appropriately diluted original bacterial suspensions were spread onto the surface of 2216E solid agar medium containing 1.0% (w / v) skim milk. After aseptic air drying, the samples were incubated upside down at 25°C for 4 days. Single colonies producing a clear zone were streaked and purified at least three times on the same skim milk agar medium to obtain protease-producing single colonies. The purified single colonies were inoculated into 5 ml of 2216E liquid agar medium and incubated on a shaker at 25°C and 180 rpm for 48 h. 600 μl of the bacterial suspension was then mixed with 200 μL of 60% glycerol and stored in a cryovial at -80°C. More than 100 protease-producing bacteria were isolated by plate (primary screening), and then fermented in a shaker (secondary screening). The protease activity of the fermentation broth was measured, and E1-8 was the strain with the highest protease production.

[0048] Example 2: Identification of Bacillus subtilis desert subspecies E1-8

[0049] 16S rDNA sequencing was performed on strain E1-8, and the sequences were compared with those of Bacillus inaquosorum TKM1, T7, NL1, GBWHF-98, J21, and J5. The similarity was over 99% in all cases. Figure 1 The selected strain underwent genome sequencing. The resulting genome sequence is indexed in NCBI under the genomic ID JAXOUB000000000. BLAST alignment analysis of the sequencing results with existing genome sequences of *Bacillus inaquosorum* in the NCBI database revealed that E1-8 showed 98.75% genomic similarity to previously reported *Bacillus inaquosorum* genomes (KCTC 13429, GOA 0031484151) and 98.29% similarity to *Bacillus inaquosorum* genomes (SI3, GOA 032463625.1). Therefore, strain E1-8 was ultimately identified as *Bacillus inaquosorum* and named *Bacillus inaquosorum* E1-8.

[0050] The selected Bacillus subsp. deserte E1-8 was deposited on October 30, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20232071, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province. The requesting institution for deposit was Qingdao Agricultural University.

[0051] Example 3: Fermentation method and parameter optimization of Bacillus subtilis desert subsp. E1-8

[0052] To explore the most suitable fermentation conditions for the production of extracellular proteases by Bacillus subtilis desert subspecies E1-8, and to improve the yield of extracellular proteases for industrial production applications, this example involves the following screening of fermentation media and optimization of pH and fermentation temperature.

[0053] The Bacillus subtilis desert subsp. *substantia* fermentation broth mentioned in this invention is a supernatant obtained by centrifugation to remove the bacterial cells. The main component of this fermentation broth is E1-8 protease, which mainly includes two extracellular proteases: serine protease and metalloproteinase. For ease of description in different scenarios, the E1-8 protease mentioned below is equivalent to the Bacillus subtilis desert subsp. *substantia* fermentation broth or simply fermentation broth.

[0054] 3.1 Effects of different carbon sources on protease production in fermentation broth of strain E1-8

[0055] The initial culture medium consisted of 0.2% yeast extract, 0.3% casein, 0.5% gelatin, and 0.3% artificial sea salt, and the pH was adjusted to 8.0.

[0056] By changing the carbon source to low-cost sweet potato, rice bran, mung bean, wheat bran (added at 2%), or high-cost yeast powder, xanthan gum, sucrose, sorbitol (added at 1%), and incubating at 37℃ with shaking at 180 r / min, the protease activity was measured, and sweet potato was selected as the optimal carbon source. Figure 2 A).

[0057] Explanation: Fermentation broth is a complex system. Generally, protease activity is used to represent the protease yield in the fermentation broth. The conversion relationship between the two is something that those skilled in the art should understand. Therefore, this descriptive method will be used throughout the following text.

[0058] 3.2 Effects of different nitrogen sources on protease production in the fermentation broth of strain E1-8

[0059] By changing the nitrogen source to low-cost soybean meal, peanut meal, fish meal (added at 2%), or high-cost casein, gelatin, soy protein, pea protein, peptone, or skim milk (added at 1%), and incubating at 37℃ with shaking at 180 r / min, the protease activity was measured to select soybean meal as the optimal nitrogen source. Figure 2 B).

[0060] 3.3 Effects of different carbon source and nitrogen source addition amounts on protease yield in fermentation broth of strain E1-8

[0061] Based on the above screening results for carbon and nitrogen sources, the effects of carbon and nitrogen source addition amounts on the protease production of strain E1-8 were further investigated.

[0062] Using 2% soybean meal as the nitrogen source, different amounts of sweet potato were added, and the optimal carbon source addition was determined to be 3%. Figure 2 C). Similarly, using 2% sweet potato as the carbon source, the optimal carbon source addition amount was determined to be 1% ( Figure 2 C).

[0063] 3.4 Effects of different fermentation pH and fermentation temperature on protease yield in fermentation broth of strain E1-8

[0064] Using 3% sweet potato as the carbon source and 1% soybean meal as the nitrogen source, the culture medium was adjusted to different pH values, and the optimal fermentation pH of 7 was selected. Figure 2 D). Furthermore, the optimized culture medium was fermented at different temperatures, and the optimal fermentation temperature was determined to be 37℃. Figure 2 D).

[0065] 3.5 Orthogonal experiment to screen the optimal fermentation medium and culture conditions

[0066] Based on the single-factor experiments, the four single factors of carbon source content, nitrogen source content, pH, and temperature were further optimized through orthogonal experiments. The experiment adopted a 4-factor, 3-level L9(3) experiment. 4 Design (Table 1). Strain E1-8 was inoculated into nine different culture media and cultured with shaking at 180 rpm. The protease activity in the fermentation supernatant was then measured. Figure 3 A). Orthogonal experimental results showed that the optimal fermentation medium and conditions were 3.5% sweet potato as the carbon source, 0.5% soybean meal as the nitrogen source, fermentation temperature of 37℃, and pH of 7.0 (Table 1). Compared with the initial medium, the optimized medium reduced costs. Furthermore, the selected optimal fermentation medium and conditions were validated; under the optimal fermentation conditions, the protease activity reached a maximum of 321.48 U / ml. Figure 3 B).

[0067] Table 1: Orthogonal experimental design and results of fermentation culture medium.

[0068]

[0069]

[0070] Example 4: Identification of extracellular protease species in fermentation broth of Bacillus subtilis desert subsp. E1-8

[0071] To further determine the types of extracellular proteases in the fermentation broth of strain E1-8, we analyzed the effects of four protease inhibitors on enzyme activity: PMSF (a specific inhibitor of serine proteases), op (a specific inhibitor of metalloproteinases), E64 (a specific inhibitor of cysteine ​​proteases), and PepA (a specific inhibitor of aspartic proteases).

[0072] The experimental results are shown in Table 2. PMSF inhibited the extracellular protease activity of strain E1-8 by 80.53%, indicating that the strain can secrete serine proteases. op inhibited the extracellular protease activity of strain E1-8 by 41.6%, indicating that the strain can secrete metalloproteinases. However, PepA and E64 had no significant effect on the extracellular protease activity of strain E1-8. This suggests that the extracellular proteases secreted by strain E1-8 are mainly composed of serine proteases and metalloproteinases (Table 2).

[0073] Table 2: Effects of protease inhibitors and metal ions on the extracellular protease activity of strain E1-8.

[0074]

[0075] Note: Table 2. a The protease and inhibitor were mixed and incubated at 4°C for 60 min. Using casein as a substrate, the protease activity was measured at 25°C and pH 7.5. The activity of the protease without inhibitor was used as a control. b ND indicates that the inhibition of protease activity is not significant.

[0076] Genome sequencing of strain E1-8 and proteomic analysis of its fermentation broth revealed two extracellular proteases, GE000323 and GE000819, which are serine proteases and metalloproteinases, respectively, accounting for 82.16% and 17.84% of the total proteases, respectively (Table 3). Figure 3 B). The nucleotide sequence of the serine protease is shown in SEQ ID NO.1 of the sequence listing, the amino acid sequence of the serine protease is shown in SEQ ID NO.2 of the sequence listing, the nucleotide sequence of the metalloproteinase is shown in SEQ ID NO.3 of the sequence listing, and the amino acid sequence of the metalloproteinase is shown in SEQ ID NO.4 of the sequence listing.

[0077] Table 3: Protease data detected by the proteome (secretome).

[0078]

[0079] Example 5: Preparation method and parameter optimization of enzymatic hydrolysate from Bacillus subtilis fermentation broth 5.1 Preparation method of enzymatic hydrolysate from Bacillus subtilis fermentation broth

[0080] 1. Prepare fresh fish skin or pig skin; soybean protein powder (protein content ≥90%, purchased from Linyi Shansong Biological Products Co., Ltd.); pea protein powder (protein content ≥80%, purchased from Yantai Dongfang Protein Technology Co., Ltd.). Chop and pre-treat the sample. The pre-treatment involves homogenizing the sample by weighing the washed and chopped sample, adding an appropriate amount of sterile water, and homogenizing on ice.

[0081] 2. Add fermentation broth (E1-8 protease) for hydrolysis. Set the hydrolysis parameters as follows: Plant-derived soybean protein: pH 12.0, 60℃, 60min, 800U / g (fermentation broth / soybean protein); Plant-derived pea protein: pH 11.0, 60℃, 50min, 800U / g (fermentation broth / pea protein).

[0082] Animal-derived fish skin: pH 9.0, 50℃, 50min, 1000U / g (fermentation broth / fish skin); Animal-derived pig skin: pH 11.0, 60℃, 50min, 600U / g (fermentation broth / pig skin).

[0083] 3. Thermal deactivation at 90℃ for 15 minutes.

[0084] 4. Centrifuge at 12000 rpm for 30 min to obtain the supernatant.

[0085] 5. Vacuum freeze-drying yields the enzymatic hydrolysis product of Bacillus subtilis fermentation broth. Preferably, it is a protein peptide freeze-dried powder.

[0086] 5.2 Parameter optimization of the preparation method of enzymatic hydrolysis products of Bacillus subtilis fermentation broth

[0087] To optimize the enzymatic hydrolysis activity of Bacillus subtilis fermentation broth for different protein substrates, we tested its optimal enzymatic hydrolysis parameters, including reaction temperature, reaction pH, enzyme-to-substrate ratio (E / S), and reaction time.

[0088] Experimental results showed that the optimal reaction temperature for this enzyme was 60℃ when the substrate was a homogenate of soybean protein, pea protein, and pig skin; and 50℃ when the substrate was a homogenate of fish skin. Figure 5 A).

[0089] This enzyme tolerates a wide pH range, exhibiting activity against all four substrates from pH 3.0 to 12.0. The optimal reaction pH is 12.0 when using soybean protein as a substrate; 11.0 when using pea protein and pig skin homogenate as substrates; and 9.0 when using fish skin homogenate as substrates. Figure 5 B).

[0090] Under optimal reaction temperature and pH conditions, the optimal enzyme-to-saturation ratio (E / S) and optimal reaction time for different substrates were determined. The optimal E / S was 800 U / g for soybean protein and pea protein; 1000 U / g for fish skin homogenate; and 600 U / g for pig skin homogenate. Figure 5 C). The amount of enzymatic hydrolysates from soybean protein, pea protein, fish skin homogenate, and pig skin homogenate increased with increasing hydrolysis time, reaching a maximum at 1 hour. Beyond 1 hour, the amount of hydrolysates did not increase significantly. Therefore, the optimal hydrolysis time was 1 hour. Figure 5 D).

[0091] Example 6: Physicochemical Properties Analysis of Enzymatic Hydrolysates from Bacillus subtilis Fermentation Broth

[0092] Based on the optimization results of the above enzymatic hydrolysis parameters, we have preliminarily established a process for enzymatic hydrolysis of plant-derived and animal-derived proteins from Bacillus subtilis fermentation broth, and prepared soybean protein hydrolysate, pea protein hydrolysate, porcine skin protein hydrolysate, and fish skin protein hydrolysate. To characterize these four protein hydrolysates, we analyzed the molecular weight distribution of peptides and the composition and content of amino acids in the hydrolysates.

[0093] The molecular weight distribution of four protease hydrolysates was determined by high performance liquid chromatography (Table 4). Figure 6 The results showed that the molecular weights of soybean protein hydrolysate, pea protein hydrolysate, fish skin protein hydrolysate, and pig skin protein hydrolysate were mainly distributed in the range of 180–2000 Da, equivalent to 2–20 peptides (peptides of 2–20 amino acids), accounting for 89.9%, 89.09%, 84.38%, and 77.51% of the total, respectively.

[0094] Table 4: Molecular weight distribution of peptides obtained from enzymatic hydrolysis of animal and plant protein substrates by crude enzyme solution of B. inaquosorum E1-8.

[0095]

[0096] The amino acid composition and content of the enzymatic hydrolysates are shown in Table 5. Seventeen amino acids were identified in these hydrolysates, including seven essential amino acids for humans: threonine, valine, methionine, isoleucine, leucine, phenylalanine, and lysine. This suggests that these hydrolysates have the potential to be developed into functional foods. Furthermore, the plant protein hydrolysates were rich in glutamic acid, accounting for 9.63% of soybean hydrolysates and 8.89% of pea hydrolysates. The most abundant amino acid in the animal protein hydrolysates was glycine, accounting for 14.31% and 14.29% of fish skin hydrolysates and pig skin hydrolysates, respectively.

[0097] Table 5. Composition and content of amino acids in animal and plant protease hydrolysates.

[0098]

[0099] Note: Table 3. * Essential amino acids

[0100] Example 7: Antioxidant activity of enzymatic hydrolysate from Bacillus subtilis fermentation broth

[0101] To evaluate the antioxidant activity of enzymatic hydrolysates of animal / plant proteins obtained from the fermentation broth of *Bacillus subtilis* (desert subsp. *subtilis*), we determined their DPPH free radical scavenging activity. First, DPPH was dissolved in a small amount of anhydrous ethanol, and then a 100 μM DPPH solution was prepared with 50% (v / v) ethanol. The solution was prepared fresh and stored in the dark. Soybean, pea, fish skin, and porcine skin hydrolysates were prepared at concentrations of 1 mg / ml, 5 mg / ml, 10 mg / ml, and 20 mg / ml, respectively. 1 ml of each sample was added to a test tube, and 2 ml of DPPH solution was added. After thorough mixing, the mixture was incubated at 25°C in the dark for 40 min, and the absorbance at 525 nm was measured. A blank zeroing group, a background absorption group, and a blank control group were also set up. The blank zeroing group used an equal volume of distilled water and 50% ethanol mixture; the background absorption group used an equal volume of 50% ethanol instead of the DPPH solution; and the blank control group used an equal volume of distilled water instead of the sample. The results are as follows Figure 7 As shown in the AD diagram, the DPPH scavenging rate of the four animal and plant protease hydrolysates increased with increasing concentration. Specifically, at a concentration of 20 mg / ml, the DPPH scavenging rate of soybean hydrolysate reached 84.12%, pea hydrolysate reached 83.98%, fish skin hydrolysate reached 75.57%, and porcine skin hydrolysate reached 58.64%. These results indicate that the animal and plant protease hydrolysates prepared from strain E1-8 protease possess strong free radical scavenging capabilities, and their excellent antioxidant potential can be applied in the cosmetics and food industries.

[0102] like Figure 7 As shown in Figure E, a comparison of the DPPH scavenging rates of the undiluted fermentation supernatant with four 20 mg / ml concentrations of enzymatic hydrolysates reveals that the DPPH scavenging rate of the fermentation supernatant is lower than that of soybean protein hydrolysate, pea protein hydrolysate, and fish skin protein hydrolysate, but slightly higher than that of porcine skin protein hydrolysate. This may be because the culture medium contains soybean meal, and the plant protein peptides released from the enzymatic hydrolysis of soybean meal have antioxidant activity. However, when the fermentation supernatant was diluted 2-fold or 5-fold, DPPH scavenging was not detected.

[0103] Example 8: Analysis of the antifreeze ability of enzymatic hydrolysate from Bacillus subtilis fermentation broth

[0104] The heat flow and freezing point of four protease hydrolysate solutions were determined by differential scanning calorimetry (DSC).

[0105] Soybean protein hydrolysate solutions, pea protein hydrolysate solutions, fish skin protein hydrolysate solutions, and porcine skin protein hydrolysate solutions were prepared at concentrations of 10 mg / ml, 20 mg / ml, and 50 mg / ml, respectively. The negative control was PBS buffer (pH 7.5), and the positive control was glycerol solution (0.5 M). The solutions were cooled from 20°C to -25°C at a rate of 5°C / min in a DSC system (TAQ2000), held at -25°C for 5 min, and then cooled at 5°C / min. / The solution was heated to 10°C at a rate of min, and heat flux was monitored during both heating and cooling. The starting point of the heat flux curve during heating was determined as the freezing point of the protease hydrolysate solution.

[0106] DSC experimental results showed that, compared with PBS and glycerol solutions, the melting peaks of soybean protein hydrolysate, pea protein hydrolysate, fish skin protein hydrolysate, and porcine skin protein hydrolysate solutions were smaller, indicating that these four plant and animal protein hydrolysate solutions inhibited ice formation and had better antifreeze properties than glycerol. Figure 8 AD). With increasing concentration, the freezing points of the four animal and plant protease hydrolysate solutions gradually decreased. Figure 9 (AD) further illustrates its antifreeze ability and its potential use as a low-temperature preservative.

[0107] In addition, we tested the freeze resistance of the Bacillus subtilis desert subsp. fermentation broth. Compared with PBS and glycerol solutions, the undiluted fermentation broth had a smaller melting peak, indicating that it inhibited ice formation and had better freeze resistance than glycerol. Compared with 20 mg / ml soybean protein hydrolysate, 10 mg / ml pea protein hydrolysate, 10 mg / ml fish skin protein hydrolysate, and 10 mg / ml porcine skin protein hydrolysate, the melting peak of the undiluted fermentation broth was not significantly different, indicating that its freeze resistance was comparable to that of the four animal and plant protein hydrolysate solutions (10-20 mg / ml). Figure 8 E). After the fermentation broth was diluted 2, 5, and 10 times, its freezing point increased significantly with increasing dilution factor. Figure 9 E) indicates that the antifreeze properties of the fermentation supernatant decrease significantly with dilution. In conclusion, the original fermentation broth can be used as a low-temperature preservative, but the application prospects of diluted fermentation broth as a low-temperature preservative are not high.

[0108] Example 9: Application of enzymatic hydrolysate from Bacillus subtilis fermentation broth in fruit preservation 9.1 Application of enzymatic hydrolysate from Bacillus subtilis fermentation broth in preventing oxidative browning of fresh-cut fruits

[0109] Sliced ​​fresh fruit is prone to oxidation, leading to browning, which is a major challenge in the storage and preservation of fresh fruit. This study investigated the effects of four protease hydrolysates on the color change of freshly cut apple slices and analyzed their practical application in preventing browning in fruits and vegetables. Fresh apples were selected and cut into slices approximately 3 mm thick. Soy protease hydrolysate solutions at concentrations of 1 mg / ml, 5 mg / ml, 10 mg / ml, and 20 mg / ml, pea protease hydrolysate solutions, fish skin protease hydrolysate solutions, and pig skin protease hydrolysate solutions were sprayed onto the surface of the fruit slices, respectively. PBS (pH 7.5) was used as a control. The slices were placed in clean petri dishes and stored at room temperature. The color change of the apple slices was observed and photographed every 24 hours. The results are as follows: Figure 10 As shown in AC, the color change mainly occurred in the first 48 hours, and different concentrations of protease hydrolysates all significantly inhibited browning of apple slices. With increasing protease hydrolysate concentration, the degree of browning in apple slices gradually decreased. Soy protease hydrolysates, pea protease hydrolysates, fish skin protease hydrolysates, and pig skin protease hydrolysates, at a concentration of 1 mg / ml, effectively inhibited browning of apple slices within 24 hours; at a concentration of 10 mg / ml, they effectively inhibited browning of apple slices within 48 hours. Figure 10 As shown in -D, the stock solution had a slight effect on preventing browning in apple slices, but when diluted 2 or 5 times, it had almost no effect on preventing browning.

[0110] Furthermore, the changes in the browning index of fresh-cut fruits were detected using a colorimeter, and the results are as follows: Figure 11 As shown, when stored at 4°C, compared to the control group, the browning index of the soybean protein hydrolysate, pea protein hydrolysate, fish skin protein hydrolysate, and pig skin protein hydrolysate treatment groups decreased by 22.15%, 16.89%, 18.30%, and 14.81%, respectively. Figure 11 A). When stored at 25°C, compared to the control group, the browning index of the soybean protein hydrolysate, pea protein hydrolysate, fish skin protein hydrolysate, and pig skin protein hydrolysate treatment groups decreased by 12.61%, 11.21%, 14.46%, and 12.92%, respectively. Figure 11B). The above results indicate that the prepared proteolytic enzyme has the potential to inhibit fruit browning.

[0111] 9.2 Application of enzymatic hydrolysate from Bacillus subtilis fermentation broth in preventing freezing damage and discoloration of fresh-cut fruits

[0112] Bananas are tropical fruits, and while refrigeration can extend their shelf life, it also makes them more susceptible to color and shape changes. This study investigated the effects of enzymatic hydrolysates on fresh-cut fruits stored under refrigeration and analyzed their practical application in the low-temperature preservation of fruits and vegetables. Bananas were sliced ​​into approximately 3mm thin slices. Soy protein peptide solutions at concentrations of 1mg / ml, 5mg / ml, 10mg / ml, and 20mg / ml, pea protein peptide solutions, fish skin collagen peptide solutions, and pig skin collagen peptide solutions were sprayed onto the surface of the fruit slices, respectively. The slices were placed in clean petri dishes and stored at 4°C. Color changes were observed and photographed every 24 hours. PBS (pH 7.5) was used as a negative control, and 0.5M glycerol as a positive control.

[0113] The morphological changes of banana slices from different treatment groups after 7 days of storage are as follows: Figure 12 As shown, different concentrations of protease hydrolysates significantly inhibited color changes and freezing damage in refrigerated banana slices. With increasing hydrolysate concentration, the degree of color and morphological changes in banana slices gradually decreased. Soybean, pea, fish skin, and pig skin hydrolysates, at a concentration of 20 mg / ml, effectively inhibited morphological changes in banana slices for 7 days. Figure 12 Browning index of banana slices from different treatment groups after 7 days of storage: Figure 13 As shown, compared with the control group, the browning index of the soybean protein hydrolysate, pea protein hydrolysate, fish skin protein hydrolysate, and pig skin protein hydrolysate treatment groups decreased by 9.41%, 5.19%, 10.09%, and 13.03%, respectively. Figure 13 The above results indicate that the prepared proteolytic hydrolysate has the potential to inhibit changes in the morphology and color of bananas.

[0114] In conclusion, all four of the above-mentioned enzymatic hydrolysis products can be used as preservatives for fresh-cut fruits.

[0115] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A strain of Bacillus subtilis desert subspecies ( Bacillus inaquosorum E1-8, characterized in that, The preservation number of Bacillus subtilis desert subspecies E1-8 is CCTCC NO:M 20232071.

2. A fermentation method for Bacillus subtilis desert subsp. E1-8 as described in claim 1, characterized in that, The fermentation medium includes the following components: 0.5% to 5% carbon source, 0.5% to 5% nitrogen source; pH 6 to 8; fermentation temperature 28℃ to 37℃; and shaking culture at 180 r / min. The carbon source of the fermentation medium is any one of sweet potato, rice bran, mung bean, wheat bran, yeast powder, xanthan gum, sucrose, and sorbitol; the nitrogen source is any one of soybean meal, peanut meal, fish meal, casein, gelatin, soy protein, pea protein, peptone, and skim milk.

3. A fermentation broth of Bacillus subtilis desert subsp. E1-8, characterized in that, It is prepared by the Bacillus subtilis desert subspecies E1-8 as described in claim 1 and the fermentation method as described in claim 2.

4. The fermentation broth according to claim 3, characterized in that, The fermentation broth contains two extracellular proteases: serine protease and metalloprotease.

5. An enzymatic hydrolysis product of Bacillus subtilis desert subsp. E1-8 fermentation broth, characterized in that, Obtained by enzymatic hydrolysis of plant-derived and / or animal-derived protein substrates from the fermentation broth as described in claim 3.

6. The fermentation broth enzymatic hydrolysis product according to claim 5, characterized in that, The preparation method of enzymatic hydrolysis products of Bacillus subtilis desert subsp. E1-8 fermentation broth includes the following steps: S1: Prepare plant-derived and / or animal-derived protein substrates and perform pretreatment. The pretreatment involves homogenizing the sample by weighing the washed and chopped sample, adding an appropriate amount of sterile water, and homogenizing on ice. S2: Add the fermentation broth as described in claim 3 for hydrolysis, and set the hydrolysis parameters; S3: Thermal deactivation at 90℃ for 15 min; S4: Centrifuge at 12000 rpm for 30 min to obtain the supernatant; S5: Vacuum freeze-drying yields the enzymatic hydrolysis product of Bacillus subsp. desertis E1-8 fermentation broth.

7. The fermentation broth enzymatic hydrolysis product according to claim 6, characterized in that, In step S2, the hydrolysis parameters for plant-derived products are: pH 11.0~12.0, 60℃, 50~60 min, fermentation broth 800 U / g; and the hydrolysis parameters for animal-derived products are: pH 9.0~11.0, 50℃~60℃, 50 min, fermentation broth 600~1000 U / g.

8. The application of the fermentation broth enzymatic hydrolysis product as described in any one of claims 5-7 in the preservation of fresh-cut fruits.

9. The application according to claim 8, characterized in that, The application for preserving fresh-cut fruit includes preventing browning of fresh-cut fruit.

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