A method for preparing a fat substitute using a pediococcus acidilactici exopolysaccharide

Abstract

The application discloses a method for preparing a fat substitute by using Pediococcus acidilactici S1 exopolysaccharide, and belongs to the technical field of fat substitute preparation, and specifically comprises the following steps: freezing and drying egg white powder and Pediococcus acidilactici S1 exopolysaccharide, then dissolving and mixing the two kinds of freeze-dried powder, and water-bath heating to induce the formation of Pediococcus acidilactici S1 exopolysaccharide-egg white protein gel. The Pediococcus acidilactici S1 exopolysaccharide-egg white protein gel is very similar to the texture of animal fat, and can be used as a fat substitute of animal fat. After the animal fat is replaced by the fat substitute in equal amount, the taste and texture of the prepared sausage product are basically completely the same as those of the original sausage product. The health degree is significantly improved. The healthy polysaccharide product is used to replace the animal fat. The fat substitute is not easy to be oxidized and has environmental stability, and has physiological activities such as anti-tumor, improvement of intestinal microecology of human bodies, anti-biofilm, adsorption of soluble heavy metals, and the like, and meets the requirements of current healthy food.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fat substitute preparation, and particularly relates to a method for preparing a fat substitute by using Streptococcus lactis S1 exopolysaccharide. BACKGROUND

[0002] The gelation property of meat emulsion products is an important functional characteristic affecting the products, and determines the quality of the products such as texture, water retention, color and the like. The traditional sausages and other related meat emulsion products are endowed with special taste and flavor due to the gelation and texture of animal fat, and the animal fat is mainly composed of saturated higher fatty acid glyceride and unsaturated higher fatty acid glyceride, and the content of the saturated higher fatty acid glyceride is higher. These fatty acids are directly related to diseases such as high blood pressure and high blood fat, and therefore, the fat substitute is one of the trends of future food development.

[0003] The fat substitute refers to a substance used to replace fat in food, can imitate the function and sensory characteristics of fat, is a substance without health risks, and the fat and calories should be significantly reduced. It can be divided into single fat substitute (divided into fat matrix type fat substitute, protein matrix type fat substitute and polysaccharide matrix type fat substitute according to different substrates) and composite fat substitute. However, single protein and polysaccharide fat mimetic cannot have all these ideal properties at the same time. The use of protein alone or polysaccharide alone has various functional and sensory limitations, such as poor mouthfeel and poor stability to salt, pH, heat treatment, freeze-thawing and the like. Protein-polysaccharide complexes can form microparticles similar in size and shape to fat globules and emulsion droplets, enabling them to imitate fat, so that they can partially or completely replace fat in food. Fat substitutes based on the combination of protein and polysaccharide have been used to replace fat in food, thereby producing low-fat food. Due to their interaction, these complexes have been shown to have better functionality than each ingredient used alone. Due to the high molecular weight and numerous functional groups of these biopolymers, they can also effectively interact with water and other molecules, thereby promoting the stability of the system. Through the interaction between polysaccharides and proteins and between proteins and proteins, a three-dimensional network structure with viscoelasticity is formed, which endows the meat products with a smooth and rich mouthfeel, and improves the texture and other qualities of the meat products.

[0004] Egg white proteins (EWP) are widely used in food production due to their excellent processing properties. Gelation is one of the most important properties of EWP, and thus EWP is often applied as a gelling agent, edible film and fat substitute in various food products. The formation of heat-induced gels of EWP is the result of protein denaturation, aggregation and coagulation. However, heat treatment techniques often result in a protein aggregation rate that is greater than the unfolding rate, thereby promoting the formation of insoluble aggregates and turbid gels, which often do not meet consumer demands in terms of gel properties.

[0005] It has been confirmed in the literature that polysaccharide-egg white protein composite gels applied to meat products can enhance the viscosity and water holding capacity of the meat product gels, but they have not been made into fat substitutes. The main reason is that the texture and performance of the prepared gels are not as good as real fat. The present application uses a self-prepared exopolysaccharide of Pediococcus acidilactici S1, which has a special structure and egg white protein to make a gel substitute for fat, and can develop new meat products, which is one of the important solutions to develop healthy meat products. SUMMARY

[0006] The present application uses a new exopolysaccharide of Pediococcus acidilactici S1 mixed with egg white protein to obtain an exopolysaccharide of Pediococcus acidilactici S1-egg white protein mixture. The texture, shape and color of the exopolysaccharide of Pediococcus acidilactici S1-egg white protein mixture are very similar to animal fat, and the same amount of exopolysaccharide of Pediococcus acidilactici S1-egg white protein mixture can be used to replace animal fat in food preparation, thereby reducing calorie intake and increasing healthy ingredients while maintaining the original flavor.

[0007] The present application provides a method for preparing a fat substitute using an exopolysaccharide of Pediococcus acidilactici, comprising the following steps:

[0008] ① Egg white protein lyophilized powder is dissolved with deionized water to obtain a 6-10% egg white protein solution. Then, 0.2% exopolysaccharide of Pediococcus acidilactici S1 is added according to the mass / volume percentage, and the pH is adjusted to 7.8-8.2 to obtain an exopolysaccharide of Pediococcus acidilactici S1-egg white protein mixture;

[0009] ② The exopolysaccharide of Pediococcus acidilactici S1-egg white protein mixture is heated at 85-95°C for 25-40 min to induce gel formation, and then immediately cooled in an ice water bath at 0±1°C to obtain an exopolysaccharide of Pediococcus acidilactici S1 fat substitute.

[0010] Further, the structural formula of the exopolysaccharide of Pediococcus acidilactici S1 is as follows: Figure 9 .

[0011] Further, the preparation method of the egg white protein freeze-dried powder is as follows: after the fresh eggs are shelled, the yolk, egg white membrane and tie are separated, and the impurities in the egg white liquid are removed. The egg white is dispersed by a high-speed disperser at 2000-5000 r / min for 4-8 min to obtain a natural and uniform egg white liquid, and the egg white protein freeze-dried powder is obtained after freeze-drying.

[0012] Further, the preparation method of the Pediococcus acidilactici S1 exopolysaccharide is as follows: the activated and passaged Pediococcus acidilactici S1 bacterial liquid is inoculated into MRS broth medium, and fermentation culture is carried out in a 37℃ constant temperature incubator, and the culture time is 36-72h; after the Pediococcus acidilactici S1 fermentation liquid culture is completed, the bacteria and other precipitable impurities are removed by centrifugation, pre-cooled anhydrous ethanol is added, and the mixture is placed in a 4℃ environment for standing, and the polysaccharide precipitate is collected; a proper amount of deionized water is added to dissolve the polysaccharide precipitate, 8-12% (m / v) trichloroacetic acid solution is added to the dissolved polysaccharide precipitate, and the mixture is placed in a 4℃ environment for standing for 10-15h, and then centrifugation is carried out to remove the protein; anhydrous ethanol is added to the supernatant again, and the polysaccharide precipitate is collected again after standing. The newly collected polysaccharide precipitate is dissolved with deionized water, and then the solution is loaded into a dialysis bag and dialyzed in deionized water at 4℃ for 2d to obtain the Pediococcus acidilactici S1 exopolysaccharide; and the powder-like Pediococcus acidilactici S1 exopolysaccharide is obtained by freeze-drying after purification.

[0013] Further, the Pediococcus acidilactici S1 is preserved in the China Center for Type Culture Collection, the preservation number is CCTCC NO: M2021229, the Latin name is Pediococcus acidilactici strain S1, the address is Wuhan University, Wuhan, Hubei, China, the postcode is 430072, and the telephone number is 027-68754052. Beneficial effects

[0014] The Pediococcus acidilactici S1 adopted in the application is isolated from the laboratory-made bacon in January 2020, and it can produce a brand-new exopolysaccharide. Figure 9 .

[0015] It is found that the polysaccharide is different from traditional plant polysaccharides in structure, and the egg white protein is combined with the polysaccharide by using a heat-induced gel technology, so that a white gel is produced, and the structure of the gel is very similar to that of pig fat; the optimal hardness of the prepared new composite gel is 223.46 g, and the water holding capacity is 94.67%. In addition, the structure of the composite gel is more compact and smooth.

[0016] When the ordinary sausage is prepared by using the fat substitute prepared by the application, the texture, taste, color and aroma of the sausage are basically unchanged, and the health level is significantly improved, the content of saturated and unsaturated higher fatty acid glycerides is significantly reduced, and healthy polysaccharide products are taken instead, the Pediococcus acidilactici S1 exopolysaccharide contains a large amount of antioxidant active substances, the prepared fat substitute has the effect of not being easily oxidized, improves the environmental stability of the fat substitute, and has physiological activities such as anti-tumor, improving intestinal microecology of human body, anti-biofilm, adsorbing soluble heavy metals, and meets the requirements of current healthy food. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Appearance diagram of EPS-EWP with different EPS addition amounts.

[0018] Figure 2 Influence diagram of different EPS addition amounts on the texture characteristics of the fat substitute.

[0019] Figure 3 Influence diagram of different EPS addition amounts on the rheological properties of the fat substitute.

[0020] Figure 4 Influence diagram of different EPS addition amounts on the water holding capacity of the fat substitute.

[0021] Figure 5 Influence diagram of different EPS addition amounts on the microstructure of the fat substitute.

[0022] Figure 6 Influence diagram of different EPS addition amounts on the molecular force of the fat substitute.

[0023] Figure 7 Influence diagram of different fat substitute addition amounts on the texture of the sausage.

[0024] Figure 8 FTIR spectrum and secondary structure peak area ratio diagram of different fat substitute sausages.

[0025] Figure 9 Structural formula of Pediococcus acidilactici S1 exopolysaccharide. DETAILED DESCRIPTION

[0026] In this embodiment, the Pediococcus acidilactici S1 exopolysaccharide used is the exopolysaccharide of the Pediococcus acidilactici S1 exopolysaccharide, and its structural formula is shown in Figure 9 .

[0027] Pediococcus acidilactici S1, preservation number CCTCC NO: M2021229, preserved in China Center for Type Culture Collection on March 15, 2021, Latin name: Pediococcus acidilactici strainS1, Address: Wuhan University, Wuhan, Hubei, China, Postcode: 430072, Phone: 027-68754052.

[0028] The purification method of P. acidipiscis S1 exopolysaccharide is as follows: the dried crude exopolysaccharide is crushed and treated with ethanol solution overnight for defatting and decolorizing. The dried residue is hot water extracted at 60°C with a solid-liquid ratio of 1:10 for 4 h. The precipitate is re-extracted as described above (2-3 times), and the supernatant is combined. The extracted solution is concentrated, precipitated with 4 volumes of anhydrous ethanol at 4°C, and the crude water extract is obtained. Then the dried crude water extract is dissolved in water, deproteinized by Sevage method, sequentially removed of fat with petroleum ether reagent, removed of pigment with macroporous resin AB-8, dialyzed against water (3000 Da), concentrated, and freeze-dried to obtain the crude polysaccharide. The crude polysaccharide is applied to a DEAE-cellulose column (26 mm x 400 mm) and eluted with distilled water at 4 mL / min, then with 0.1, 0.2 M and 0.3 M NaCl. Each fraction is collected and the carbohydrate content is determined by Sanshu Biotech at 490 nm by phenol-sulfuric acid method. The main polysaccharide fraction is collected, concentrated, and dialyzed against distilled water (3000 Da) for 48-72 h. Then the polysaccharide solution is applied to a Sephacryl S-400 HR column (26 mm x 1000 mm) and eluted with distilled water at a flow rate of 1.0 mL / min, and monitored by phenol-sulfuric acid method to obtain the purified P. acidipiscis S1 exopolysaccharide.

[0029] Methods for determining various indicators in this example

[0030] a: texture

[0031] To evaluate the suitability of the gel for many food applications, the P. acidipiscis S1 exopolysaccharide was added to egg white protein solution, and the hardness and elasticity of the gel sample were determined according to the method described by Ma et al. (Ma Y, Shan A, Wang R, et al. Characterization of egg white powder gel structure and its relationship with gel properties influenced by pretreatment with dry heat [J]. Food Hydrocolloids, 2021, 110.) The hardness and elasticity of the composite gel are shown in Table 2. Figure 2 , Figure 2 It is shown that the hardness and elasticity of the composite gel reach the maximum value when the P. acidipiscis S1 exopolysaccharide addition amount is 0.6%.

[0032] b: Determination of dynamic rheology

[0033] According to the method of Mirarab Razi et al. (Mirarab Razi S, Motamedzadegan A, Shahidi A, et al. The effect of basil seed gum (BSG) on the rheological and physicochemical properties of heat-induced egg albumin gels [J]. Food Hydrocolloids, 2018, 82: 268-277), the strain sweep was first performed at 1 Hz and 0.01-10% strain using a rheometer to determine the linear viscoelastic region (LVR). On this basis, the frequency sweep was performed at 1% strain amplitude from 0.1 to 25 Hz, and three parallel measurements were performed. The dynamic rheological graph of the composite gel prepared under different Pediococcus acidilactici S1 exopolysaccharide addition amounts is shown in Figure 2. Figure 3 Anvari M et al. (Anvari M, Chung D. Dynamic rheological and structural characterization of fish gelatin - Gum arabic coacervate gels cross-linked by tannic acid [J]. Food Hydrocolloids, 2016, 60: 516-524) considered that when the storage modulus (G') of the coacervate is all higher than the loss modulus (G'), it indicates that the coacervate has a high elastic gel-like structure. According to Masoomeh Raei et al. (Rheological and structural characteristics of whey protein-pectin complex coacervates [J]. Journal of food engineering, 2018, 228 (jul.): 25-31.), it is considered that heating will make the protein molecules unfold, and in the presence of EPS, a complex structure is formed, which promotes the cross-linking between proteins and can increase the modulus value. According to Figure 3 A shows the effect of different EPS addition amounts on the apparent viscosity of EPS-EWP composite gel. The increase of EPS addition amount increases the apparent viscosity of EPS-EWP composite gel. The dynamic frequency sweep of EWP and EPS-EWP composite gel is shown in Figure 2B. Figure 3B-C. The loss modulus (G") and elastic modulus (G') curves of EWP and EPS-EWP composite gel were similar. The composite G' and G" increased with the increase of EPS addition amount, and both were G'>G", indicating that the formed was elastic gel. tan δ>1 mainly showed viscosity, while tan δ<1 was mainly considered as elastic characteristics. As shown in Fig. 2D, all values of tan δ were between 0.1 and 1.0, indicating that the EPS-EWP composite gel was an elastic gel. It can also be seen that the gel had good elastic characteristics when the EPS addition amount was in the range of 0.2-0.8%. Figure 3 D, tan δ of all values were between 0.1 and 1.0, indicating that the EPS-EWP composite gel was an elastic gel. It can also be seen that the gel had good elastic characteristics when the EPS addition amount was in the range of 0.2-0.8%.

[0034] c: determination of water holding capacity

[0035] According to the method of Mirarab Razi et al. (Mirarab Razi, S., Motamedzadegan, A., Shahidi, A, et al. The effect of basil seed gum (BSG) on the rheological and physicochemical properties of heat-induced egg albumin gels [J]. Food Hydrocolloids, 2018, 82, 268-277), 15 g of gel sample was placed in a centrifuge tube and centrifuged at 6000 x g for 20 min in a centrifuge. The mass of the gel before and after centrifugation was weighed, and the water holding capacity WHC of the gel was the percentage of the mass ratio before and after centrifugation. Each sample was determined in triplicate and the average value was taken. The water holding capacity of the composite gel prepared under different P. acidipropionici S1 exopolysaccharide addition amounts is shown in Fig. 2C. Figure 4 According to Figure 4 , the water holding capacity was the largest (94.67%) when the P. acidipropionici S1 exopolysaccharide addition amount was 0.6%, which was significantly increased by 18.83% (p<0.05) compared with the egg white protein gel without P. acidipropionici S1 exopolysaccharide addition

[0036] d: microstructure (SEM)

[0037] Figure 5Freeze-dried samples of composite gels were cut with a razor blade according to the method of Xue et al. (Xue H, Tu Y, Zhang G, et al. Mechanism of ultrasound and tea polyphenol assisted ultrasound modification of egg white protein gel [J]. Ultrason Sonochem, 2021, 81: 105857) and fixed on SEM column with double-sided conductive tape, then gold-plated using Model IB-3 ion sputtering instrument. The samples were placed in a scanning electron microscope (Hitachi S-4800, Japan) to observe at an acceleration voltage of 3 kV at 4000 times magnification, and the atlas was collected using XT Microscope Control software. Scanning electron micrographs of composite gels with different amounts of Pediococcus acidilactici S1 exopolysaccharide are shown in Figure 6. Figure 5 Gel samples with EEP in the range of 0.2%-0.8% showed a highly cross-linked protein matrix and complex network structure, with a smoother, more uniform and denser structure.

[0038] e. Determination of intermolecular forces

[0039] According to the method described by Wu et al. (Wu D, Guo J, Wang X, et al. The direct current magnetic field improved the water retention of low-salt myofibrillar protein gel under low temperature condition [J]. LWT, 2021, 151: 112034) with slight modifications. Gel samples (1 g) were mixed with S1 (0.6 M NaCl) and homogenized for 1 min. The homogenized solution was left to stand at 4°C for 1 h, and the supernatant was saved. The obtained precipitate was mixed with S2 (1.5 M urea + 0.6 M NaCl) and homogenized, then centrifuged at 10000 x g at 4°C for 10 min, and the supernatant was saved. The obtained precipitate was mixed with S3 (8 M urea + 0.6 M NaCl), S4 (0.5 M β-mercaptoethanol + 0.6 M NaCl + 8 M urea) in turn, homogenized, and centrifuged, and all the obtained supernatants were saved. Finally, the protein content in the supernatants was measured by the biuret method. Each sample was measured in triplicate. The solubility in S1, S2, S3 and S4 represents the contribution of ionic bonds, hydrogen bonds, hydrophobic interactions and disulfide bonds, respectively. The effect of different EPS additions on the molecular forces of the gels is shown in Figure 7.Figure 6 When the EPS addition amount is 0.2-0.8%, the protein molecules are expanded by heat, more hydrophobic groups are exposed, and covalent cross-linking is formed between the EPS and the EWP, promoting the stability of the protein molecular structure. Example 1

[0040] A method for preparing a fat substitute from Streptococcus lactis S1 exopolysaccharide, the specific steps of which are as follows:

[0041] Step 1: Preparation of chicken egg white protein freeze-dried powder

[0042] After the fresh eggs are shelled, the yolk, egg white membrane and ligament are separated, and the impurities in the egg white liquid are removed. The egg white is dispersed with a high-speed disperser at 3000 r / min for 6 min to obtain a natural and uniform egg white liquid, which is then freeze-dried and stored at 4°C for standby use.

[0043] Step 2: Preparation of Streptococcus lactis S1 exopolysaccharide freeze-dried powder

[0044] The activated and passaged Streptococcus lactis S1 bacterial solution is inoculated into MRS broth medium at an inoculation amount of 2% (v / v) and fermented in a 37°C constant temperature incubator for 48 h. After the Streptococcus lactis S1 fermentation broth is cultured, the bacteria and other precipitated impurities are removed by centrifugation, 3 times the volume of pre-cooled anhydrous ethanol is added, and the mixture is placed in a 4°C refrigerator for standing. The polysaccharide precipitate is collected. An appropriate amount of deionized water is added to dissolve the polysaccharide precipitate. After the polysaccharide precipitate is dissolved, 10% (m / v) trichloroacetic acid solution is added, and the mixture is placed in a 4°C refrigerator overnight, and then centrifuged to remove the protein. 3 times the volume of anhydrous ethanol is added to the supernatant, and the mixture is allowed to stand again to collect the polysaccharide precipitate. The newly collected polysaccharide precipitate is dissolved with deionized water, and then placed in a dialysis bag and placed in a 4°C refrigerator. The dialysis bag is dialyzed with deionized water for 2 days to obtain Streptococcus lactis S1 exopolysaccharide. The purified exopolysaccharide is freeze-dried at -50°C to obtain a powder of Streptococcus lactis S1 exopolysaccharide.

[0045] Step 3: Preparation of Streptococcus lactis S1 exopolysaccharide fat substitute

[0046] The egg white protein freeze-dried powder is dissolved with deionized water to obtain an 8% egg white protein solution. Then, 0.2% Streptococcus lactis S1 exopolysaccharide (w / v, calculated based on the volume of the egg white protein solution) is added while stirring, the pH is adjusted to 8.0 with citric acid, and the Streptococcus lactis S1 exopolysaccharide-egg white protein mixture is heated in a water bath at 90°C for 30 min to induce gel formation. Immediately after heating, the mixture is cooled in an ice water bath, the beaker is sealed with plastic wrap, and a Streptococcus lactis S1 exopolysaccharide fat substitute is obtained, which is stored at 4°C for standby use.

[0047] The hardness and elasticity of the fat substitute formed in this example were 119.77 g and 0.68 g, respectively; the water holding capacity was 87.00%; the storage modulus increased, the intermolecular force was strengthened, the fat substitute formed a complex network structure, and the microstructure was uniform with smaller voids. Example 2

[0048] A method for preparing a fat substitute from Pediococcus acidilactici S1 exopolysaccharide, which comprises the following specific steps:

[0049] Step 1: Preparation of egg white protein freeze-dried powder

[0050] After the fresh eggs were shelled, the yolk, egg white membrane and ligament were separated, and the impurities in the egg white liquid were removed. The egg white was dispersed with a high-speed disperser at 3000 r / min for 6 min to obtain a natural and uniform egg white liquid, which was then stored at 4°C after freeze-drying for standby use.

[0051] Step 2: Preparation of Pediococcus acidilactici S1 exopolysaccharide freeze-dried powder

[0052] The activated and passaged Pediococcus acidilactici S1 bacterial solution was inoculated into MRS broth medium at an inoculation amount of 2% (v / v), and then fermented in a 37°C constant temperature incubator for 48 h. After the fermentation of the Pediococcus acidilactici S1 culture was completed, the bacterial bodies and other precipitable impurities were removed by centrifugation, 3 times the volume of pre-cooled anhydrous ethanol was added, and the mixture was placed in a 4°C refrigerator for standing. The polysaccharide precipitate was collected. An appropriate amount of deionized water was added to dissolve the polysaccharide precipitate, and then 10% (m / v) trichloroacetic acid solution was added to the solution, which was placed in a 4°C refrigerator overnight, and then centrifuged to remove the protein. 3 times the volume of anhydrous ethanol was added to the supernatant, and after standing, the polysaccharide precipitate was collected again. The newly collected polysaccharide precipitate was dissolved with deionized water, and then placed in a dialysis bag and placed in a 4°C refrigerator. The dialysis bag was dialyzed with deionized water for 2 days to obtain Pediococcus acidilactici S1 exopolysaccharide. The crude polysaccharide was purified and freeze-dried at -40°C to obtain a powder of Pediococcus acidilactici S1 exopolysaccharide.

[0053] Step 3: Preparation of Pediococcus acidilactici S1 exopolysaccharide fat substitute

[0054] The egg white protein freeze-dried powder was dissolved with deionized water to obtain an 8% egg white protein solution. Then, 0.4% Pediococcus acidilactici S1 exopolysaccharide (w / v, calculated based on the volume of the egg white protein solution) was added while stirring, the pH was adjusted to 8.0 with citric acid, and the Pediococcus acidilactici S1 exopolysaccharide-egg white protein mixture was heated in a 90°C water bath for 30 min to induce gel formation. Immediately after stopping heating, the beaker was cooled in an ice water bath, and the beaker was sealed with plastic wrap to obtain a Pediococcus acidilactici S1 exopolysaccharide fat substitute, which was stored at 4°C for standby use.

[0055] The hardness and elasticity of the fat substitute formed in this example are 161.53 g and 0.79 g, respectively; the water holding capacity is 89.67%; the storage modulus increases, and the intermolecular force is strengthened; the fat substitute forms a complex network structure, and the microstructure is uniform and dense. Example 3

[0056] A method for preparing a fat substitute from Pediococcus acidilactici S1 exopolysaccharide, which comprises the following specific steps:

[0057] Step 1: Preparation of egg white protein freeze-dried powder

[0058] After the fresh eggs are shelled, the yolk, egg white membrane and ligament are separated, and the impurities in the egg white liquid are removed. The egg white is dispersed with a high-speed disperser at 5000 r / min for 4 min to obtain a natural and uniform egg white liquid, which is stored at 4°C after freeze-drying for standby use.

[0059] Step 2: Preparation of Pediococcus acidilactici S1 exopolysaccharide freeze-dried powder

[0060] The activated and passaged Pediococcus acidilactici S1 bacterial solution is inoculated into MRS broth medium at an inoculation amount of 5% (v / v), and fermented in a 37°C constant temperature incubator for 72 h. After the fermentation of the Pediococcus acidilactici S1 culture is completed, the bacteria and other precipitated impurities are removed by centrifugation, 3 times the volume of pre-cooled anhydrous ethanol is added, and the mixture is placed in a 4°C refrigerator for standing. The polysaccharide precipitate is collected. An appropriate amount of deionized water is added to dissolve the polysaccharide precipitate. After the polysaccharide precipitate is dissolved, 12% (m / v) trichloroacetic acid solution is added, and the mixture is placed in a 4°C refrigerator for 10 h, and then centrifuged to remove the protein. 3 times the volume of anhydrous ethanol is added to the supernatant, and the polysaccharide precipitate is collected again after standing. The newly collected polysaccharide precipitate is dissolved with deionized water, and then placed in a dialysis bag and placed in a 4°C refrigerator. The dialysis bag is dialyzed with deionized water for 2 d to obtain Pediococcus acidilactici S1 exopolysaccharide. The purified Pediococcus acidilactici S1 exopolysaccharide is freeze-dried at -18°C to obtain a powder.

[0061] Step 3: Preparation of Pediococcus acidilactici S1 exopolysaccharide fat substitute

[0062] The egg white protein freeze-dried powder is dissolved with deionized water to obtain a 6% egg white protein solution. Then, 0.6% Pediococcus acidilactici S1 exopolysaccharide (w / v, calculated based on the volume of the egg white protein solution) is added while stirring, the pH is adjusted to 8.2 with citric acid, and the Pediococcus acidilactici S1 exopolysaccharide-egg white protein mixture is heated in a water bath at 85°C for 40 min to induce gel formation. Immediately after stopping heating, the mixture is cooled in an ice water bath, and the beaker is sealed with plastic wrap. A Pediococcus acidilactici S1 exopolysaccharide fat substitute is obtained, which is stored at 4°C for standby use.

[0063] The hardness and elasticity of the fat substitute formed by the present embodiment are 223.46 g and 0.82 g respectively; the water holding capacity is 94.67%; the storage modulus is the largest, the intermolecular force is stronger, the fat substitute forms a complex network structure, and the microstructure is smooth, uniform and dense. Example 4

[0064] A preparation method of a fat substitute prepared from Pediococcus acidilactici S1 exopolysaccharide, and the specific steps are as follows:

[0065] Step 1: Preparation of egg white protein freeze-dried powder

[0066] After the fresh eggs are shelled, the yolk, egg white membrane and tie are separated, and the impurities in the egg white liquid are removed. The egg white is dispersed with a high-speed disperser at 2000 r / min for 8 min to obtain a natural and uniform egg white liquid, which is stored at 4℃ after freeze-drying for standby use.

[0067] Step 2: Preparation of Pediococcus acidilactici S1 exopolysaccharide freeze-dried powder

[0068] The activated and passaged Pediococcus acidilactici S1 bacterial solution is inoculated into MRS broth medium at an inoculation amount of 1% (v / v), and fermented in a 37℃ constant temperature incubator for 36 h. After the fermentation of the Pediococcus acidilactici S1 culture is completed, the bacteria and other precipitated impurities are removed by centrifugation, 3 times the volume of pre-cooled anhydrous ethanol is added and placed in a 4℃ refrigerator for standing, and the polysaccharide precipitate is collected. The polysaccharide precipitate is dissolved by adding an appropriate amount of deionized water, and after it is dissolved, 8% (m / v) trichloroacetic acid solution is added, and placed in a 4℃ refrigerator for 15 h, and then centrifuged to remove the protein. 3 times the volume of anhydrous ethanol is added to the supernatant again, and after standing, the polysaccharide precipitate is collected again. The newly collected polysaccharide precipitate is dissolved with deionized water, and then placed in a dialysis bag and placed in a 4℃ refrigerator, and dialyzed with deionized water for 2 d to obtain Pediococcus acidilactici S1 exopolysaccharide. After purification, the powder is freeze-dried at -30℃ to obtain Pediococcus acidilactici S1 exopolysaccharide powder.

[0069] Step 3: Preparation of Pediococcus acidilactici S1 exopolysaccharide fat substitute

[0070] The egg white protein freeze-dried powder is dissolved with deionized water to obtain a 10% egg white protein solution. Then, 0.8% Pediococcus acidilactici S1 exopolysaccharide (w / v, calculated based on the volume of the egg white protein solution) is added while stirring, the pH is adjusted to 7.8 with citric acid, and the Pediococcus acidilactici S1 exopolysaccharide-egg white protein mixture is heated in a water bath at 95℃ for 25 min to induce gel formation. Immediately after stopping heating, ice water bath cooling is performed, the beaker is sealed with plastic wrap, and a Pediococcus acidilactici S1 exopolysaccharide fat substitute is obtained, which is stored at 4℃ for standby use.

[0071] Results and analysis of index determination

[0072] The hardness and elasticity of the fat substitute formed in this embodiment are 162.49 g and 0.72 g respectively; the water holding capacity is 87.13%; the storage modulus is slightly reduced, the intermolecular force is weakened, the fat substitute forms a complex network structure, and the microstructure is uniform with smaller voids.

[0073] A method for preparing a fat substitute from Pediococcus acidilactici S1 exopolysaccharide, comprising the following specific steps:

[0074] After the fresh eggs are shelled, separate the yolk, egg white membrane and ligament, and remove the impurities in the egg white liquid. Use a high-speed disperser to stir and disperse the egg white at 3000 r / min for 6 min to obtain a natural and uniform egg white liquid, which is stored at 4°C after freeze-drying for standby.

[0075] The egg white protein freeze-dried powder is dissolved with deionized water to obtain an 8% egg white protein solution. Then, citric acid is used to adjust the pH to 8.0, and heating at 90°C for 30 min in a water bath is used to induce gel formation. Immediately after stopping heating, ice water bath cooling is performed, the beaker is sealed with plastic wrap, and is stored at 4°C for standby.

[0076] The hardness and elasticity of the gel formed in this embodiment are 101.55 g and 0.37 g respectively; the water holding capacity is 79.67%; the storage modulus is low, the intermolecular force is weak; a complex network structure is formed, the microstructure has larger voids and a rough surface, and it cannot be used as a fat substitute. Example 5

[0077] A method for preparing low-fat pork sausage using the fat substitute prepared in Example 1, comprising the following specific steps:

[0078] Traditional sausages (control group) are prepared using lean meat, pork back fat, ice, salt, and sodium tripolyphosphate, and the fat substitute prepared in Example 1 is used to replace pork back fat of the same mass to prepare low-fat sausages.

[0079] The lean meat and pork back fat are broken by a meat chopper, and the EPS-EWP composite gel is broken into gel particles by a homogenizer for 2 min; the pork back fat and fat substitute particles are mixed, 5 g of ice water is added, and gel emulsion is formed by homogenization at 3000 rpm for 2 min; the lean meat, gel emulsion, remaining ice water, and other ingredients are stirred and emulsified by a silent cutter.

[0080] The meat batter is filled into pork casing using a sausage stuffer. The sausages are cooked in water at 90°C for 30 min. Cooking loss is determined after each batch of sausages is cooked. Then, the remaining sausages are vacuum packaged and stored in a refrigerator at 4°C until analysis. Three independent batches of sausages are prepared on different days. In addition, three sausages are included in each batch for each determination.

[0081] Table 1 lists the textural characteristics of sausages prepared by replacing pork back fat with composite gel particles. Compared with the control group (replacement ratio 0%), the sausages in the replacement group showed increased firmness and chewiness. p <0.05), with minimal changes in elasticity, cohesiveness, and resilience. Texture parameters reached their highest values ​​at substitution ratios of 87.5% and 93.75%. Chen et al. achieved higher firmness and chewiness in Frankfurt sausages prepared with pre-emulsified rapeseed oil and porcine plasma protein. Compared to animal fat, the gel has smaller particles, allowing for efficient filling into the three-dimensional gel network matrix of meat proteins. Furthermore, appropriate fat substitutes can improve meat gels and increase firmness by fostering more protein-protein interactions during cooking. During sausage cooking, heat-induced unfolding of protein structures leads to the exposure of active groups, which promotes interactions between proteins and muscle proteins in the gel, forming a fine gel network. Figure 7 The results accurately reflect the cross-sections of sausages with different substitution ratios, with the smoothest cross-section observed when the substitution ratio is 93.75%.

[0082] Table 1: Texture characteristics of sausages prepared by replacing pork back fat with composite gel particles

[0083] Substitution ratio / % Hardness / g Elasticity Coherency Chewiness Resilience 0 5197.98 ± 629.71 b ]] 0.92 ± 0.04 b ]] 0.84 ± 0.04 a ]] 4047.93 ± 690.9 b ]] 0.48 ± 0.22 ab ]] 50 5294.54 ± 318.80 b ]] 0.96 ± 0.00 a ]] 0.88 ± 0.03 a ]] 4516.62 ± 443.0 ab ]] 0.52 ± 0.27 a ]] 75 5545.26 ± 463.30 ab ]] 0.95 ± 0.20 ab ]] 0.84 ± 0.02 a ]] 4366.36 ± 302.7 ab ]] 0.42 ± 0.07 b ]] 87.5 6124.70 ± 163.71 a ]] 0.95 ± 0.13 ab ]] 0.86 ± 0.03 a ]] 4781.37 ± 89.79 a ]] 0.51 ± 0.12 a ]] 93.75 6143.57 ± 93.96 a ]] 0.94 ± 0.02 ab ]] 0.88 ± 0.05 a ]] 4865.70 ± 42.73 a ]] 0.51 ± 0.13 a ]] 100 5428.21 ± 141.95 b ]] 0.95 ± 0.01 ab ]] 0.85 ± 0.01 a v]]> 4457.51 ± 184.39 ab ]] 0.50 ± 0.20 a ]]

Claims

1. A method for preparing a fat substitute using Pediococcus acidilactici exopolysaccharide, characterized by: It comprises the following steps: ① The egg white protein freeze-dried powder is dissolved with deionized water to obtain a 6-10% egg white protein solution; 0.2% of the exopolysaccharide of Pediococcus acidilactici S1 is added according to the mass / volume percentage, and the pH is adjusted to 7.8-8.2, to obtain a Pediococcus acidilactici S1 exopolysaccharide-egg white protein mixture; ② The Pediococcus acidilactici S1 exopolysaccharide-egg white protein mixture is heated at 85-95℃ for 25-40 min to induce gel formation, and then cooled in an ice water bath at 0±1℃ immediately after heating is stopped, to obtain a Pediococcus acidilactici S1 exopolysaccharide fat substitute; The structural formula of the Pediococcus acidilactici S1 exopolysaccharide is as follows: ； The Pediococcus acidilactici S1 is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2021229.

2. The method of preparing a fat substitute using Pediococcus acidilactici exopolysaccharide according to claim 1, characterized by: The preparation method of the egg white protein freeze-dried powder is as follows: after the fresh eggs are shelled, the yolk, egg white membrane and chalaza are removed, and the impurities in the egg white liquid are removed; the egg white is dispersed by a high-speed disperser at 2000-5000 r / min for 4-8 min to obtain a natural and uniform egg white liquid, which is freeze-dried to obtain the egg white protein freeze-dried powder.

3. The method of preparing a fat substitute using Pediococcus acidilactici exopolysaccharide according to claim 1, characterized by: The preparation method of the Pediococcus acidilactici S1 exopolysaccharide is as follows: the activated and passaged Pediococcus acidilactici S1 bacterial liquid is inoculated into MRS broth medium and fermented in a 37℃ constant temperature incubator for 36-72 h; after the fermentation of the Pediococcus acidilactici S1 is completed, the bacteria and other precipitable impurities are removed by centrifugation, pre-cooled anhydrous ethanol is added and placed in a 4℃ environment, and the polysaccharide precipitate is collected; a proper amount of deionized water is added to dissolve the polysaccharide precipitate, 10% (m / v) trichloroacetic acid solution is added after the polysaccharide precipitate is dissolved, and the mixture is placed in a 4℃ environment for 10-15 h, and then centrifuged to remove the protein; anhydrous ethanol is added to the supernatant again, and the polysaccharide precipitate is collected again after standing; the newly collected polysaccharide precipitate is dissolved with deionized water, and then dialyzed in a 4℃ environment with deionized water, to obtain the crude exopolysaccharide of Pediococcus acidilactici S1; after purification, the powder is dried by a freeze dryer to obtain the powder-like exopolysaccharide of Pediococcus acidilactici S1.

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