Method for preparing emulsion gel using Bacillus subtilis and application of emulsion gel

Abstract

The present invention belongs to the field of bioengineering fermentation technology, and specifically provides a method for preparing an emulsion gel using bacillus subtilis and the application of the emulsion gel, the method comprising the steps of: expanding and culturing bacillus subtilis SNBS-43; uniformly mixing a fully hydrated soy protein isolate solution and soybean oil in a certain proportion to obtain a soy protein isolate emulsion; inoculating bacillus subtilis SNBS-43 into the soy protein isolate emulsion, fermenting, and obtaining an emulsion gel prepared using bacillus subtilis. The present invention studies the properties, protein secondary structure changes, and fat replacement ability of the emulsion gel prepared by fermentation of bacillus subtilis SNBS-43, and develops it to replace fat, such as the fat in meat products such as beef, meatballs, and chicken sausage, which can achieve the purpose of a healthy diet and provide a new way for industrially preparing foods with low fat content and no loss of taste.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering fermentation, and specifically provides a method for preparing an emulsion gel by utilizing Bacillus subtilis and application of the emulsion gel. Background Art

[0002] Bacillus subtilis is a probiotic widely used in traditional fermented soy products, including soybean paste, fermented bean curd, fermented black beans, and natto. It exhibits strong enzyme-producing abilities. Observations have shown that fermented soybean paste produces a viscous substance, fermented bean curd solidifies, and natto develops stringy textures. These viscous and coagulated structures enhance the food's texture. However, limited research has examined the coagulation effects of Bacillus subtilis fermentation. Summary of the Invention

[0003] In order to solve the above technical problems, the present invention provides a method for preparing an emulsion gel using Bacillus subtilis and applications of the emulsion gel.

[0004] The present invention is achieved by providing a method for preparing an emulsion gel using Bacillus subtilis, wherein the Bacillus subtilis is Bacillus subtilis SNBS-43, and the method comprises the following steps:

[0005] 1) Expanding the culture of Bacillus subtilis SNBS-43;

[0006] 2) uniformly mixing the fully hydrated soy protein isolate solution and soybean oil in a certain proportion to obtain a soy protein isolate emulsion;

[0007] 3) inoculating Bacillus subtilis SNBS-43 into the soy protein isolate emulsion and fermenting the emulsion to obtain an emulsion gel prepared by using Bacillus subtilis.

[0008] Preferably, Bacillus subtilis SNBS-43 (Bacillus subtilis) has been deposited by the General Microbiology Center of the China Culture Collection Administration on September 26, 2021, with the deposit number CGMCC No. 23482.

[0009] More preferably, step 1) comprises the following steps:

[0010] 101) Thaw the Bacillus subtilis SNBS-43 stored in glycerol at -80°C in a freezer, streak a certain amount of the bacterial suspension onto an LB solid medium plate, and culture overnight at 37°C to form colonies;

[0011] 102) Pick a colony with an inoculation loop, inoculate it into LB liquid culture medium, and culture it at 37°C and 150 rpm for 24 hours.

[0012] More preferably, in step 2), the fully hydrated soy protein isolate solution and soybean oil are mixed in a certain proportion to prepare an O / W soy protein isolate emulsion with a concentration of 8% and an oil phase of 10%, and the homogenization condition is: 12000 rpm for 3 min.

[0013] More preferably, in step 3), the inoculation amount of Bacillus subtilis SNBS-43 is 4% of the soy protein isolate emulsion.

[0014] Further preferably, in step 3), the soy protein isolate emulsion inoculated with Bacillus subtilis SNBS-43 is shaken evenly, placed in a constant temperature incubator at 37°C, fermented until a gel is formed, and transferred to a 4°C refrigerator for storage for 12 hours to form a complete gel, that is, an emulsion gel prepared using Bacillus subtilis.

[0015] The present invention also provides an emulsion gel, which is prepared according to the above method for preparing the emulsion gel using Bacillus subtilis.

[0016] The present invention also provides the application of the emulsion gel, which is added into meat products as a fat substitute.

[0017] Preferably, in the above application, the amount of the emulsion gel added is 5-20% of the meat product.

[0018] More preferably, the meat product is meatballs or sausages.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] The present invention studies the properties, protein secondary structure changes, and fat replacement capacity of the emulsion gel prepared by fermentation of Bacillus subtilis SNBS-43. The emulsion gel can be developed to replace fat in meat products such as beef, meatballs, and chicken sausage, achieving the goal of healthy diet and providing a new way to industrially prepare foods with low fat content without sacrificing taste. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the growth curve of Bacillus subtilis SNBS-43 of the present invention;

[0022] Figure 2 Schematic diagram of the protease hydrolysis cycle of Bacillus subtilis SNBS-43 of the present invention;

[0023] Figure 3 is the acid production curve of Bacillus subtilis SNBS-43 of the present invention;

[0024] Figure 4 This is the free thiol content curve of the emulsion gel prepared by fermentation of Bacillus subtilis SNBS-43 of the present invention;

[0025] Figure 5 This is the turbidity change curve of the emulsion gel prepared by fermentation of Bacillus subtilis SNBS-43 of the present invention;

[0026] Figure 6 This is the water holding capacity change curve of the emulsion gel prepared by fermentation of Bacillus subtilis SNBS-43 of the present invention;

[0027] Figure 7 This is a bar graph of the intermolecular forces of the emulsion gel prepared by fermentation of Bacillus subtilis SNBS-43 of the present invention;

[0028] Figure 8 This is a bar graph showing the effects of different emulsion gel addition amounts on the cooking loss of meatballs;

[0029] Figure 9 This is a bar graph showing the effect of different emulsion gel addition amounts on the water holding capacity of meatballs;

[0030] Figure 10 This is a bar graph showing the effects of different amounts of emulsion gel added on the surface hydrophobicity of meatballs;

[0031] Figure 11 The figure is a graph showing the effect of different emulsion gel addition amounts on the pH value of minced meat. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0033] The culture medium and its formula used in the embodiment of the present invention are as follows:

[0034] LB medium: 10 g sodium chloride, 10 g tryptone, 5 g yeast extract powder, 1000 mL distilled water, pH 7.2; 20 g agar (solid medium).

[0035] Protease identification medium: 115g skim milk powder, 20g agar, add distilled water to 1L.

[0036] The present invention is described in detail below with reference to specific embodiments:

[0037] Example 1: Determination of the growth curve of Bacillus subtilis SNBS-43 strain

[0038] Thaw the strain stored in glycerol stock at -80°C, streak a certain amount of the bacterial solution onto LB solid medium plates, and incubate at 37°C overnight. Pick a colony with an inoculation loop and inoculate it into LB liquid medium. Incubate at 37°C and 150 rpm for 24 hours before use.

[0039] The cultured Bacillus subtilis SNBS-43 was inoculated into LB liquid medium at a 2% inoculum size and cultured at 37°C and 150 rpm for 24 hours. Samples were taken every 2 hours to measure the OD value at 600 nm and a growth curve of Bacillus subtilis SNBS-43 was plotted.

[0040] Results: As attached Figure 1 As shown, it was found that Bacillus subtilis SNBS-43 reached the logarithmic phase in 6-15h and entered the plateau phase in 15-20h. It was found that the bacteria grew vigorously around 15h of culture.

[0041] Example 2: Determination of protease activity of Bacillus subtilis SNBS-43

[0042] Inoculate the activated Bacillus subtilis strain SNBS-43 into LB liquid medium and incubate at 37°C, 180 rpm, and shake for 24 hours. Centrifuge at 10,000 rpm and 4°C for 15 minutes, and collect the supernatant. Use a microporator to evenly punch holes in a sterilized protease identification medium plate. Inject 100 μL of the supernatant into each well and incubate in a 37°C incubator for 24 hours.

[0043] Results: As attached Figure 2 As shown, a smooth hydrolysis zone appeared around the bacterial solution of Bacillus subtilis SNBS-43, and the ratio of the protease hydrolysis zone to the colony area was 6.8±0.03, proving that it has the ability to produce protease.

[0044] Example 3: Determination of acid production capacity of Bacillus subtilis SNBS-43 strain

[0045] The test strain Bacillus subtilis SNBS-43 was inoculated into 100 mL LB liquid culture medium at an inoculum size of 2%, and cultured at 37°C and 180 rpm for 24 h. The pH of the fermentation broth was measured every 2 h to draw an acid production curve.

[0046] Results: With pH as the vertical axis and time as the horizontal axis, a pH curve was drawn (see Appendix Figure 3 ) and found that Bacillus subtilis SNBS-43 has a very weak acid-producing ability and can even raise the pH value to create an alkaline environment suitable for its own growth. Therefore, it can be basically ruled out that the fermentation of soy protein isolate by Bacillus subtilis SNBS-43 is the direct acid production of the microorganism.

[0047] Example 4: A method for preparing emulsion gel based on Bacillus subtilis SNBS-43 fermentation

[0048] Preparation of soy protein isolate solution: Dissolve 8% soy protein in distilled water and stir overnight in a magnetic stirrer until fully hydrated.

[0049] Emulsion preparation: The fully hydrated soy protein isolate solution was mixed with 10% soybean oil and homogenized at 12000×g for 3 min to form an O / W soy protein isolate emulsion with an SPI concentration of 8% and an oil phase of 10%.

[0050] Inoculation: Bacillus subtilis SNBS-43 in Example 1 was inoculated into the fully hydrated emulsion at a 4% inoculum amount.

[0051] Fermentation: Shake the inoculated emulsion evenly and place it in a constant temperature incubator at 37°C to ferment until gel is formed.

[0052] Gel formation: Store at 4°C for 12 hours to allow complete gel formation.

[0053] Prepare samples: freeze-dry the formed complete gel using a vacuum freeze dryer for later use.

[0054] Example 5: Determination of gel properties of emulsions prepared by fermentation with Bacillus subtilis SNBS-43

[0055] Determination of fermentation state and color of emulsion gel

[0056] During the fermentation preparation process, samples were taken every 2 hours and measured using a colorimeter. L* represents the color index of brightness, ranging from 0-100, where larger values ​​represent whiter colors and smaller values ​​represent darker colors. a* represents the color index of red and green, where larger positive values ​​represent redder colors and smaller negative values ​​represent greener colors. b* represents the color index of yellow and blue, where larger positive values ​​represent yellower colors and smaller negative values ​​represent bluer colors. Calculation is based on the following formula:

[0057]

[0058] Results: As shown in Table 1, as the fermentation time increases, it can be found that the gel mainly changes from liquid to solid, and the fluidity gradually decreases. After 12 hours, it is found that it will not flow when inverted, and it is completely solidified to form a self-supporting gel. The colorimeter measurement shows that the brightness L * The value decreases, and the brightness is lowest at 12 hours. As the fermentation time increases, it can be found that the gel formed after 12 hours is more red. This may be because the emulsion after homogenization is whiter than the original color of soy protein isolate. As the fermentation time increases, the biofilm produced by the fermentation metabolism of Bacillus subtilis appears red on the surface, so the phenomenon of leaning towards red appears. *The yellow-green color change during the fermentation process was not obvious. The total color difference (ΔE) showed that the gel sample had the largest color change at 12 hours of fermentation, indicating that the color changed significantly before and after the fermentation gel formed.

[0059] Table 1 Emulsion gel fermentation state and color

[0060]

[0061] Determination of thiol content in emulsion gel

[0062] The sample (5 mg) was mixed with 5.0 mL of Tris-glycine buffer for 30 min. The buffer included 8 mol / L urea, 0.09 mol / L glycine, 4 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.086 mol / LTris-hydrochloric acid (pH 8.0), and 40 μL of Ellman's solvent (5,5′-dithio-bis-2-nitrobenzoic acid, 4 mg / mL, dissolved in Tris-glycine buffer). The supernatant was collected by centrifugation at 7000 × g for 10 minutes, and the absorbance of the supernatant was measured at 412 nm. All samples were analyzed independently in triplicate using the following formula:

[0063]

[0064] Where A 412 is the absorbance at 412 nm; D is the dilution factor; C is the sample concentration, in mg / mL.

[0065] Results: Thiol content is a quantitative indicator of changes in protein structure. Figure 4 It was found that the free thiol content of the formed protein emulsion gel began to decrease with increasing fermentation degree, reaching a minimum at 12 hours of fermentation. The reduction in thiol groups may be attributed to the aggregation of protein molecules, which releases buried thiol groups in the protein molecules and oxidizes them to form disulfide bonds.

[0066] Determination of turbidity and water holding capacity (WHC) of emulsion gel

[0067] The sample was dissolved in distilled water at a concentration of 6%, and the supernatant was diluted. Turbidity was expressed as absorbance measured at 660 nm using a UV spectrophotometer (UV-2600, Japan). WHC was determined by centrifugation (8000 g, 4°C, 20 min) of a portion of the emulsion gel using the following formula:

[0068]

[0069] Where W1 is the weight of the emulsion gel after removing water, in g; W is the weight of the fermentation-induced emulsion gel, in g.

[0070] Results: By the attached Figure 5 As shown, by measuring the turbidity of the emulsion gel at different fermentation times, it was found that the turbidity gradually increased with the increase of fermentation time. It was found that the turbidity of the gel began to rise sharply around 6 hours and reached the maximum at 12 hours, indicating that the gel was formed and the turbidity was the maximum when the fermentation reached 12 hours. Gel water holding capacity is a key parameter for evaluating gel performance. WHC can reflect the ability of the food matrix to stabilize water molecules through capillary action. Water holding capacity can reflect the strength of the gel to a certain extent. Hydrogen bonding is the main force for maintaining the secondary structure of proteins, and it is also one of the reasons for maintaining the good hardness and water holding capacity of the fermented emulsion gel. The hydrogen bonding of the fermented emulsion gel is the main force, so the fermented emulsion gel has strong water holding capacity and strength. As shown in the attached figure Figure 6 As shown, the water-holding capacity of the fermented emulsion gel increased with fermentation time, reaching its maximum at 12 hours, significantly higher than at other fermentation times (P < 0.05). The strength and turbidity of the fermented emulsion gel also reached their maximum, indicating that the gel had the densest structure and the strongest water-holding capacity at 12 hours of fermentation.

[0071] Determination of intermolecular forces in emulsion gels

[0072] The solubility of the gel was measured using five types of solutions to determine the molecular driving force involved in the gelation: 0.05 mol / L NaCl (SA), 0.6 mol / L NaCl (SB), 0.6 mol / L NaCl + 1.5 mol / L urea (SC), 0.6 mol / L NaCl + 8 mol / L urea (SD), and 0.6 mol / L NaCl + 8 mol / L urea + 0.5 mol / L 2-β-mercaptoethanol (SE). The gel (2 g) was mixed with 10 mL of each solution in a magnetic stirrer at 5°C for 1 hour. The resulting homogenate was centrifuged at 20,000 × g for 15 minutes, and the protein concentration in the supernatant was determined using a BCA kit.

[0073] Results: Intermolecular interactions drive and maintain the formation of gel structure. Figure 7 As shown, the content of hydrophobic interactions and hydrogen bonds in the intermolecular interactions of fermented emulsion gels was significantly higher than that of ionic bonds, nonspecific associations, and disulfide bonds (P < 0.05). The low solubility of SA and SB may be related to the binding and aggregation of SPI under fermentation conditions. The disruption of nonspecific associations and ionic bonds that maintain the native structure of SPI leads to changes in the molecular structure of SPI. The solubility of SC and SD increased significantly with fermentation time (P < 0.05), indicating that hydrophobic interactions and hydrogen bonds are the primary drivers of fermented emulsion gels.

[0074] Example 6: Study on the Preparation of Emulsion Gel by Fermentation with Bacillus subtilis SNBS-43 to Replace Fat Determination of Meatball Texture Characteristics

[0075] The texture of meatballs containing different emulsion gels replacing fat was measured using hardness, elasticity, resilience, cohesion, adhesion, and chewiness as indicators. Hardness describes the internal bonding force required to deform or penetrate the meatball to maintain its shape. Elasticity refers to the ability of the meatball to deform under external force and return to its original state after the external force is removed. Cohesion describes the property of food that resists damage during chewing and remains tightly connected to keep the food intact. It represents the strength of the internal bonding of the meatball. Chewability reflects the sample's continued resistance to chewing, the so-called "bite", which represents the energy required to chew solid food and is the product of hardness, cohesion, and elasticity.

[0076] The texture analyzer was equipped with a 50 kg load cell and a 50 mm cylindrical probe (P / 50). Thawed samples were heated in an 80°C water bath for 5 minutes and then cooled to room temperature. Prior to TPA analysis, the samples were cut into cylinders with a diameter and height of 15 and 20 mm, respectively. Samples were measured at room temperature using front, middle, and back test speeds of 2.0, 2.0, and 5.0 mm / s, a compression distance of 10 mm, and a trigger force of 5.0 g. The meatballs were tested for hardness, springiness, cohesiveness, gummyness, and chewiness.

[0077] Table 2 Effect of adding emulsion gel on the texture of meatballs

[0078]

[0079] Texture analysis revealed that as the emulsion gel content increased, the cohesiveness, elasticity, chewiness, and gumminess of meatballs containing different amounts of EG significantly improved compared to meatballs containing 20% ​​adipose tissue. The hardness and springiness were similar to those of meatballs containing 20% ​​adipose tissue, but significantly lower than those of all-lean meat (P < 0.05). The experiments revealed that meatballs containing 15% EG exhibited significantly higher cohesiveness, elasticity, gumminess, and chewiness than those containing other amounts. However, the hardness of meatballs containing 15% EG decreased compared to other emulsion gel content, but remained higher than that of meatballs containing 20% ​​adipose tissue. This confirms that 15% EG possesses the best meatball texture.

[0080] Meatball cooking loss

[0081] Cooking loss is an important indicator used to measure the loss of meat when cooked at high temperatures. Higher cooking loss will reduce the taste, flavor and nutrition of the meat. Figure 8As shown, by measuring the cooking loss of meatballs with varying amounts of emulsion gel, it was found that as the amount of emulsion gel increased, the cooking loss initially decreased and then increased. The lowest cooking loss was achieved at 15% emulsion gel, which was significantly lower than the control group without emulsion gel (P < 0.05). Adding an appropriate amount of gel to the meat filling thoroughly homogenizes the gel surface with the minced meat, increasing the cohesiveness of the minced meat and locking in the moisture of the meatballs. This reduces the internal moisture retention caused by high-temperature heating, and increases the bonding strength between the emulsion gel and the meat, locking in the internal moisture and maintaining the juiciness of the meatballs, a key metric for evaluating meatball taste.

[0082] Weigh the same number of grams of meatballs with different amounts of emulsion gel added (W) into weighed centrifuge tubes and weigh them (W1). Cook them in an 85°C water bath for 15 minutes (until the center temperature reaches 75°C). After removing the cooking juice, weigh the cooked meatballs and centrifuge tubes again (W2). The formula is as follows:

[0083]

[0084] Where W1 is the weight of the meatballs and centrifuge tube before cooking; W2 is the weight of the meatballs and centrifuge tube before cooking; and W is the weight of the meatballs.

[0085] Results: The 20% EG group showed an increasing trend in cooking loss. This is because oil can lubricate and connect food particles, and the emulsion gel replacing part of the fat helps ensure a tight connection between the various components of the meatballs. However, when the emulsion gel content is too high, its cohesion effect is weaker than that of meatballs prepared with full fat, resulting in excessive juice loss and increased cooking loss.

[0086] Determination of water holding capacity of meatballs

[0087] The water holding capacity of meatballs is an important indicator for locking in meat juice and improving the taste. The water holding capacity of meatballs prepared by replacing fat with different emulsion gel contents was measured. Meatballs of equal mass were prepared and placed into pre-weighed centrifuge tubes (W0). The combined weight was (W1), and the mixture was centrifuged at 10,000 rpm for 10 minutes. The centrifuged water was then aspirated, and the weight after centrifugation was (W2). The formula is as follows:

[0088]

[0089] By the attached Figure 9The results of observations on the water-holding capacity of meatballs show that increasing the amount of emulsion gel added increases the water-holding capacity. At 10%, 15%, and 20% EG additions, the water-holding capacity was not significantly different from the control group, but significantly higher than that of the 5% EG group. Both low and high fat replacement levels can lead to gel structure instability, water loss, and reduced meatball juiciness, resulting in a reduced texture and flavor. The addition of 15% emulsion gel as a fat replacement effectively maintained the water-holding capacity of the meatballs, maintaining a similar level to that of the control group.

[0090] Determination of surface hydrophobicity

[0091] The surface hydrophobicity of meatballs reflects the distribution of hydrophobic amino acids on the protein surface. Properly increasing surface hydrophobicity contributes to meatball stability. Excessive increases in surface hydrophobicity can reduce protein stability, weaken protein-water interactions, and promote hydrophobic aggregation and protein denaturation, hindering the formation of a gel network structure. Therefore, measuring surface hydrophobicity is crucial.

[0092] Weigh 0.6 g of raw minced meat sample and suspend and dissolve it in 20 mL of phosphate buffer (pH 6.0). Homogenize until the minced meat is evenly distributed in the solution. Pipette 1 mL and add 100 μL of bromophenol blue. Use 100 μL of 1 mg / mL bromophenol blue added to 1 mL of phosphate buffer as a reference. Vigorously shake for 20 minutes. Centrifuge (6000 rpm, 4°C, 15 minutes). Aspirate 0.5 mL of the supernatant and add 4.5 mL of phosphate buffer. Compare the color at 595 nm with the phosphate buffer as a blank. Each treatment group was measured in parallel three times. The calculation formula is:

[0093]

[0094] Results: As attached Figure 10 As shown in the results, it was found that with the increase of the amount of latex gel added, the surface hydrophobicity of the sample increased significantly. When the amount of latex gel added was 15% and 20%, the surface hydrophobicity was significantly higher than that of the control group (P<0.05).

[0095] Changes in pH value of minced meat

[0096] The pH value reflects the acidity and flavor of meat. The acidity of raw minced meat significantly impacts meatball preparation. To investigate whether replacing fat with emulsion gel affects the pH of minced meat, the pH values ​​of minced meat with varying amounts of emulsion gel were measured. 2.5 g of minced meat sample was weighed and added to 10 mL of pre-cooled distilled water. The mixture was homogenized in a high-speed disperser and then measured using a pH meter. Once the reading stabilized, the pH was recorded. Three replicates were performed for each sample.

[0097] Results: By the attached Figure 11As shown in the results, it was found that the pH value of the minced meat decreased significantly with the increase of the emulsion gel content from 0% to 10%. The pH value of the minced meat in the 15% and 20% addition groups increased significantly, but was still lower than that of the control group. This may be because the pH value of the emulsion gel itself is acidic after fermentation, so adding it to the minced meat will lower the pH value of the minced meat. However, the pH value of the minced meat at all addition amounts is still within the normal range.

[0098] Electronic tongue measurement

[0099] The sourness, bitterness, astringency, aftertaste, umami, kokumi, and saltiness of raw meat minced meat with 0%, 5%, 10%, 15%, and 20% fat replacement and full-fat fat were measured using an electronic tongue. Using the TS-5000Z Taste Analysis System, samples containing different amounts of emulsion gel as fat replacement were diluted 1:10 with ultrapure water and centrifuged at 5000 rpm for 15 minutes. The supernatant was collected and filtered with filter paper to obtain the electronic tongue test solution. The basic tastes of sourness, bitterness, astringency, saltiness, umami, and kokumi were digitally measured. Each sample was tested in triplicate.

[0100] Results: As shown in the table below, sourness was slightly lower than that of the full-fat group at various addition levels, but kochiness was higher than the control group. Saltiness and umami were similar to those of the control group, but bitterness was slightly higher, and astringency was significantly reduced. Overall, these indicators indicate that the addition of fermented emulsion gel did not significantly alter the flavor of the raw minced meat, and even enhanced its kochiness. This suggests that fermentation does not introduce any additional flavor-degrading substances, demonstrating its ability to replace fat while maintaining both texture and flavor.

[0101] Table 3 Effect of emulsion gel addition on minced meat flavor

[0102]

[0103]

[0104] Determination of color difference

[0105] The color of meat is an important factor in evaluating the freshness of meat. Brightness (L * ) and redness (a * ) is an important indicator of meat color. The higher the myoglobin content, the greater the lean meat content, and the brighter the red. The density and content of fat particles and myofibrillar protein both affect light scattering. The specific measurement method is as follows: remove the ground beef sample from the 4°C refrigerator, remove the surface moisture, cut into small cylindrical sections, and measure three points on the cross-section using a CR-20 colorimeter. Before measuring the sample, calibrate the instrument with a white plate, and record the sample's L value, a value, and b value. Each treatment group is measured in parallel three times, and the final average is taken.

[0106] Results: As shown in the table below, as the amount of fat replaced by the emulsion gel increases, the brightness of the minced meat increases and the redness decreases. This is due to the addition of less lean meat and more protein gel. The reduced lean meat content leads to a weakening of the red color, while the brightness values ​​in the 5% EG, 10% EG and 15% EG groups did not change significantly. The brightness of the 20% EG group was significantly higher than that of the other control groups (P<0.05). The difference in brightness may be due to the different distribution and light reflectivity of smaller fat globules and larger fat particles during the treatment, indicating that the protein in the emulsion gel will affect the overall color difference of the product. The emulsion gel replaces fat and wraps around the surface of lean meat protein, neutralizing the red color of lean meat myoglobin and increasing the brightness. Yellowness value (b * ) showed a decreasing trend with increasing emulsion gel replacement, with the yellowness value in the 15% EG group significantly lower than in the 5% EG and 10% EG groups. The addition of 15% EG significantly reduced the yellowness of fat, with the reduction being most significant at 20% (P<0.05). Overall, both the 15% and 20% EG groups demonstrated significant effects in reducing yellowness and improving brightness. The redness reduction value in the 15% group was slightly lower than that in the 20% group, resulting in a more vibrant color than the 20% EG group. Therefore, the 15% EG addition level is more suitable.

[0107] Table 4 Effect of emulsion gel addition on meat color

[0108]

[0109] Sensory evaluation

[0110] The sensory analysis was conducted by a team of 12 graduate students and staff from the Department of Food Science. The meatballs were reheated in 85°C hot water until the internal temperature reached 72°C. The meatballs were then randomly distributed to a panel of 12 assessors. The meatballs were rated on a scale of 1 to 10, representing very soft to very firm, very dry to very juicy, dark to light, and not oily to very oily. All ten panelists received professional training.

[0111] Table 5 Apparent scores of emulsion gels with different addition amounts

[0112]

[0113] Results: Ten trained panelists conducted a sensory evaluation of the meatballs at 5%, 10%, 15%, and 20% replacement fat addition levels (see the table below). The average scores are plotted in Table 4. The results showed that the firmness increased with increasing addition level, the juiciness of the meatballs increased with increasing emulsion gel addition, the color became lighter, and the greasy taste decreased. Therefore, a 15% addition level scored higher and was considered the most suitable.

Claims

1. A method for preparing an emulsion gel using Bacillus subtilis, characterized in that: The Bacillus subtilis is Bacillus subtilis SNBS-43, which has been deposited by the General Microbiology Center of the China National Microorganism Culture Collection Administration on September 26, 2021, with the deposit number CGMCC No. 23482. The method comprises the following steps: 1) Expanding the culture of Bacillus subtilis SNBS-43; 2) The fully hydrated soy protein isolate solution and soybean oil were mixed in a certain proportion to prepare an O / W soy protein isolate emulsion with a soy protein isolate concentration of 8% and an oil phase of 10%. The homogenization conditions were: 12000 rpm for 3 minutes; 3) inoculating Bacillus subtilis SNBS-43 into the soy protein isolate emulsion, shaking evenly, placing it in a constant temperature incubator at 37°C, fermenting until a gel is formed, and storing it in a refrigerator at 4°C for 12 hours to form a complete gel, thereby obtaining an emulsion gel prepared using Bacillus subtilis. Specifically, the inoculation amount of Bacillus subtilis SNBS-43 is 4% of the soy protein isolate emulsion.

2. The method for preparing emulsion gel using Bacillus subtilis according to claim 1, characterized in that: Step 1) includes the following steps: 101) Thaw the Bacillus subtilis SNBS-43 stored in glycerol at -80°C in a freezer, streak a certain amount of the bacterial suspension onto an LB solid medium plate, and culture overnight at 37°C to form colonies; 102) Pick a colony with an inoculation loop, inoculate it into LB liquid culture medium, and culture it at 37°C and 150 rpm for 24 hours.

3. An emulsion gel, characterized in that The emulsion gel is prepared according to the method for preparing the emulsion gel using Bacillus subtilis according to any one of claims 1 to 2.

4. The use of the emulsion gel according to claim 3, characterized in that Added to meat products as a fat substitute.

5. The use of the emulsion gel according to claim 4, characterized in that: The added amount of the emulsion gel is 5-20% of the meat product.

6. The use of the emulsion gel according to claim 4, characterized in that: The meat product is meatballs or sausages.

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