Calcium modified bacterial cellulose and application thereof in low-salt water animal meat paste

Abstract

The invention provides calcium modified bacterial cellulose and application thereof in low-salt water livestock meat paste, and belongs to the technical field of food processing. The calcium modified bacterial cellulose is obtained by taking calcium gluconate as a carbon source and carrying out in-situ fermentation on gluconacetobacter xylinum in a liquid culture medium through an ion coordination method. According to the calcium-modified bacterial cellulose disclosed by the invention, on the premise of maintaining the flavor and texture of the water livestock meat paste product, the salt addition amount and content of the water livestock meat paste product can be reduced, and the salty taste perception and the overall quality of the water livestock meat paste can be improved. The invention provides a new strategy for developing healthier and low-salt meat paste products produced by water livestock.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and particularly relates to a calcium-modified bacterial cellulose and its application in low-salt livestock and poultry meat products. Background Art

[0002] Excessive salt intake is a major public health problem. According to the data of the World Health Organization (WHO), the average salt intake per person per day is 9 to 12 grams, which is twice the maximum recommended amount. High salt intake is the main cause of chronic diseases such as hypertension and cardiovascular diseases. Therefore, the WHO advocates reducing salt intake by 30% by 2025. Approximately 30% of the human daily salt intake comes from meat products, which is the second largest source of sodium in the diet. One of the main functions of salt in processed meat products is to dissolve myofibrillar protein (MP) and impart an ideal taste and flavor.

[0003] Currently, the sodium reduction strategies in the food industry mainly involve using salt substitutes (KCl, MgCl2 or CaCl2), changing the size, shape and distribution of salt crystals, introducing new processing technologies (ultrasound-assisted sodium tripolyphosphate), and coordinating multi-sensory effects. However, these methods often cannot completely solve the sensory and texture challenges associated with low-salt products. 70% - 95% of the sodium residues in food are not perceived. Adjusting the food matrix is an effective way to enhance the perception of saltiness. The influence of food structure on the perception of saltiness includes affecting the release rate and utilization of salt, changing the mixing efficiency of food bolus and saliva, changing the salt transport process, and affecting the spatial contact between salt and receptors. Adding biopolymers (proteins and polysaccharides) to food gels can regulate the texture and / or interact with the tongue mucosa to prolong sodium retention, thereby enhancing the perception of saltiness. Polysaccharides are macromolecules formed by the polymerization of monosaccharides and can be extracted from a variety of animals, plants and microorganisms.

[0004] Polysaccharides have a significant impact on the structure and microstructure of composite MP gels. For example, carrageenan improves the gel strength of low-salt surimi gels, promotes the formation of immobilized water, resulting in uneven spatial distribution of sodium, thereby enhancing the perception of saltiness. Carboxymethylated nanocellulose forms a dense and uniform gel network in MP, increasing gel hardness and reducing the fluidity of water. The formation of a network-reinforced MP gel by cellulose in the MP matrix depends on its shape and aspect ratio, and cellulose filling exhibits a percolation threshold. Nanocellulose, as a green functional material, exists in the form of cellulose nanocrystals, cellulose nanofibrils and bacterial cellulose (BC).

[0005] BC is a straight-chain polymer without side chains, composed of many (1-4) β-glycosidic linked glucose units. The nearly linear glucan chains form highly regular nanofibrils with nanoscale cross-sectional dimensions through intramolecular and intermolecular hydrogen bonds. BC can be synthesized by a variety of bacteria, such as Gluconacetobacter, Bordetella, and Komagataeibacter. Previous studies isolated a strain of Komagataeibacter xylinus FM883 from kombucha, and the produced bacterial cellulose mainly consisted of the α phase. BC has high purity, crystallinity, degree of polymerization, water absorption and retention capacity, tensile strength, and biocompatibility, and is suitable as a reinforcing filler for fragile food hydrogels. For example, BC enhances the juiciness and chewiness of meatballs and increases the water-holding capacity of surimi by strengthening the network structure. Although bacterial cellulose has been used to enhance the texture in food gels, its effect on the perception of saltiness in low-salt surimi has not been thoroughly explored.

[0006] Reducing the salt content in meat products weakens the myofibrillar protein network, increases the gel pore size, accelerates water loss, and reduces texture and saltiness, especially in surimi products, affecting their structure, texture, and shelf life. Summary of the Invention

[0007] To solve the problem of poor texture after salt reduction in surimi products, the present invention provides a method that can significantly reduce the salt addition in surimi products without reducing the flavor and texture of surimi products.

[0008] To achieve the above object, the inventors provided the following technical solutions:

[0009] A calcium-modified bacterial cellulose (BCCa), using calcium gluconate as a carbon source, is obtained by in-situ fermentation of Komagataeibacter xylinus in a liquid medium through an ion coordination method.

[0010] Further, the components of the liquid medium include 40-50 g / L calcium gluconate, 0.8-1.2 g / L KH2PO4, 4-6 g / L yeast extract, 10-20 g / L MgSO4, 4-6 g / L peptone, 0.8-1.2 g / L citric acid, and 1.5-2.5% ethanol (volume percentage).

[0011] Further, the calcium-modified bacterial cellulose has an alpha crystal structure with a crystallinity of 26-27%.

[0012] Further, the sodium ion diffusion coefficient of the calcium-modified bacterial cellulose is (2.39±0.98)×10 - 12 cm 2 s-1 。

[0013] A preparation method of calcium-modified bacterial cellulose. The preparation method is to add the seed liquid of Bacillus xylophilus acetic anhydride at an addition amount of 6-10% by mass percentage to a liquid culture medium, and incubate the culture at 25-35 °C for 6-8 days; soak the obtained film in an edible alcohol solution with a volume percentage of 70-80% for 2-3 days, and then boil it in pure water for 1-2 h; the bacterial concentration of the seed liquid is (3.0-4.0)×10 8 cfu / mL.

[0014] A low-salt aquatic product gel. The gel contains 80-90% of aquatic product meat paste, 1.5-2% of NaCl, 0.5-1% of calcium-modified bacterial cellulose, and the rest is water, all of which are mass percentages.

[0015] Furthermore, the preparation method of the low-salt aquatic product gel is to thaw the frozen aquatic product meat paste at 2-8 °C until the central temperature is 0-4 °C, put 80-90% of the aquatic product meat paste and ice into a meat grinder, and stir the mixture at a speed of 200-400 r / min for 8-12 min until the aquatic product meat paste becomes viscous; after adding 1.5-2% of NaCl, stir at 500-700 r / min for 2-3 min, then add 0.5-1% of calcium-modified bacterial cellulose and stir for 1-2 min, and form pellets by hand; keep it in a constant temperature water bath at 35-45 °C for 50-70 min, and keep it in a constant temperature water bath at 85-95 °C for 15-25 min to form an aquatic product meat paste gel, and immediately ice-bath it for 25-35 min to obtain the gel, and all the above percentages are mass percentages.

[0016] Furthermore, an application of calcium-modified bacterial cellulose in low-salt aquatic product meat paste products. The meat paste products include frozen meat paste products and low-temperature cooked Chinese-style fresh meat paste products, such as meatballs, meat cakes, etc.

[0017] The beneficial effects of the present invention are:

[0018] (1) The calcium-modified bacterial cellulose (BCCa) of the present invention can enhance the salty taste perception of low-salt aquatic product meat paste. BCCa can significantly promote the diffusion rate and release of sodium ions in low-salt aquatic product meat paste, and can achieve a salty taste effect equivalent to that of high salt even under low-salt conditions.

[0019] (2) The calcium-modified bacterial cellulose of the present invention improves the texture and structure of low-salt aquatic meat mince. BCCa can make the structure of myofibrillar proteins denser and more stable, transforming the secondary structure of proteins from β-turns and random coils to α-helices and β-sheets, thereby enhancing the gel strength and hardness of low-salt aquatic meat mince. The addition of BCCa makes the microstructure of aquatic meat mince more continuous, uniform, orderly, and dense, significantly improving the water-holding capacity of low-salt aquatic meat mince, reducing water loss during cooking, and improving the texture and sensory properties of the gel.

[0020] (3) The calcium-modified bacterial cellulose of the present invention can reduce the salt content of aquatic meat mince products, conforming to the trend of healthy diet. Without reducing the flavor and texture of aquatic meat mince products, BCCa can significantly reduce the salt addition amount and content in aquatic meat mince products, conforming to the healthy diet trend of reducing salt intake advocated by the World Health Organization (WHO).

[0021] (4) The present invention provides a green and efficient food additive. On the one hand, BCCa is a green functional material suitable as a reinforcing filler for fragile food hydrogels. On the other hand, the application method of BCCa is simple. Only a small amount of BCCa needs to be added during the preparation of aquatic meat mince, which can significantly improve the texture and flavor of the product, having high application value and economy. Description of the Drawings

[0022] Figure 1 For the preparation and properties of the calcium-modified bacterial cellulose described in the specific embodiments. Schematic diagram of the in-situ fermentation method for synthesizing BCCa (a); SEM and EDX element distribution maps of BC and BCCa (b); rheological analysis of BC and BCCa (c - h).

[0023] Figure 2 For the structural characterization of the calcium-modified bacterial cellulose described in the specific embodiments. WAXS spectra (a), XRD (b), FT-IR (c) of BC and BCCa; C1s (d) and Ca2p XPS spectra (e) of BCCa, 13 C NMR spectrum (f) and DG-TDG (e).

[0024] Figure 3 For the effect of the calcium-modified bacterial cellulose described in the specific embodiments on the perception of saltiness in surimi products. Effects of BCCa on the diffusion rate of Na + , Na + release (b, c), electronic tongue detection (d), saltiness (e), and umami (f).

[0025] Figure 4Effect of calcium-modified bacterial cellulose on the properties of surimi products described in the specific implementation manner. Effects of BCCa on appearance (a), flavor (b), whiteness (c), cooking loss (d), water content (e), water holding capacity (f), gel strength (g), and hardness (h).

[0026] Figure 5 Effect of calcium-modified bacterial cellulose on the water content of surimi products described in the specific implementation manner. Effects of BCCa on water distribution (a), MRI image (b), peak area (c), and peak area ratio (d).

[0027] Figure 6 Effect of calcium-modified bacterial cellulose on the structure of surimi products described in the specific implementation manner. FTIR (a), secondary structure of amide I band (b), TG (c), and XRD (d).

[0028] Figure 7 Effect of calcium-modified bacterial cellulose on the microstructure (a) and sodium chloride distribution (b) of surimi products described in the specific implementation manner. Specific implementation manner

[0029] To elaborate on the technical content, achieved objectives, and effects of the technical solution in detail, the following is described in detail with specific examples in conjunction with the accompanying drawings.

[0030] Example 1

[0031] 1 Preparation of calcium-modified bacterial cellulose (BCCa)

[0032] The strain FM883 is Komagataeibacter xylinus, which is preserved in the China Center for Type Culture Collection with the preservation number CCTCC M 2019127. The 16S rRNA gene of the strain FM883 is stored in GenBank with the accession number MW757206.

[0033] BC fermentation liquid medium: 2% glucose, 0.5% yeast extract, 0.1% K2HPO4, 1.5% MgSO4, 2% ethanol, natural pH value.

[0034] BCCa fermentation liquid medium: containing 45 g / L calcium gluconate, 1 g / L KH2PO4, 5 g / L yeast extract, 15 g / L MgSO4, 5 g / L peptone, 1 g / L citric acid, 2% ethanol (v / v).

[0035] Preparation of BC and BCCa: First, the FM883 seed solution (3.5×10 8The cfu / mL) was added to BC and BCCa liquid media at an addition amount of 8% respectively, and cultured at a constant temperature of 30 °C for 7 days. The obtained bacterial cellulose membranes (BC and BCCa) were respectively immersed in 75% edible alcohol solution for 2 days, and then boiled in pure water for 1 hour (changing water multiple times during the period). The media after taking the membranes were continued to be cultured at 30 °C to obtain the next batch of bacterial cellulose membranes until the media were consumed.

[0036] Characterization and Rheological Properties of Calcium-Modified Bacterial Cellulose

[0037] Fourier transform infrared spectroscopy (FTIR) was carried out using Thermo Fisher Scientific Nicolet iS20 (USA) and the KBr pellet method, with a wavelength range of 400 to 4000 cm -1 , and the sampling interval was 1 cm -1 . To further study the effect of introducing calcium on the hydrogen bond system of BC, the region of 3000 - 3600 cm -1 was selected for spectral fitting to evaluate the differences in the hydrogen bond systems between the two samples. First, the overlapping FTIR peaks were separated by deconvolution within the region, and the original spectra were quantified using Peakfit v4.12 software. X-ray photoelectron spectroscopy (XPS) tests were carried out using Thermo Scientific K-Alpha (Al-Kα excitation source, USA). X-ray diffraction (XRD) analysis was carried out using Empyrean Rigaku SmartLab SE (Japan). XRD was scanned between 10° and 50° (2θ) with a step interval of 0.04°. Wide-angle X-ray scattering (WAXS) data were collected using Xenocs Xeuss 2.0 (France). Scanning electron microscope (SEM) images were obtained using Hitachi Regulus 8230 (Japan). Before inspection, the samples were coated with gold (Au), and the energy-dispersive X-ray (EDX) mappings of the surface element distributions of BC and BCCa were recorded. Solid-state nuclear magnetic resonance spectroscopy was tested using the method of Brouwer and Mikolajewski (2023). According to the method of Busuioc et al. (2022) with slight modification, thermogravimetric-differential scanning calorimetry (TG-DSC) of the samples was carried out using NETZSCH STA 449F3. The samples were placed in an open crucible made of alumina, and heated from 30 °C to 800 °C at a rate of 10 °C / min under a dry air flow rate of 40 mL / min; an empty alumina crucible was used as a reference. The sample mass was approximately 21 mg.

[0038] In the creep and dynamic rheology tests, BC and BCCa were used in the hydrated state with dimensions of 2×2 cm. The tests were carried out using a rheometer (Discovery HR-10, TA Instruments, UK) equipped with 40 mm stainless steel parallel plates according to the method described by Liao et al. (2024). In the creep test, an initial shear stress of 100 Pa was applied to the sample for 120 s and then the stress was released for 180 s. Frequency sweeps were performed at a strain amplitude of 1%, and at 4 °C, the frequency decreased logarithmically from 20 to 1.0 Hz. Frequency sweeps were performed at a strain amplitude of 1%, and at 4 °C, the frequency decreased logarithmically from 20 to 1.0 Hz. The temperature sweep was from 25 °C to 90 °C at a heating rate of 2 °C / min and then cooled from 90 °C to 25 °C with a strain of 1% and 1 Hz.

[0039] Sodium ion diffusion coefficient of 3 calcium-modified bacterial cellulose

[0040] Preparation of BC / Au electrode: Accurately weigh 0.0016 g of BC, disperse it in 3 mL of ultrapure water, sonicate for 30 min, and apply 10 μL on the gold (Au) electrode (mix well before taking), and dry it with an infrared lamp to obtain the working electrode.

[0041] Preparation of BCCa / Au electrode: Accurately weigh 0.0016 g of BCCa, disperse it in 3 mL of ultrapure water, sonicate for 30 min, and apply 10 μL on the gold electrode (mix well before taking), and dry it with an infrared lamp to obtain the working electrode.

[0042] Electrochemical tests were carried out in 0.1 M NaCl solution using a three-electrode system consisting of a cellulose-modified working electrode, a platinum wire counter electrode, and an Ag / AgCl (3 M KCl) reference electrode. Alternating current impedance spectroscopy (EIS) measurement: The applied constant potential was 0.8 V, the excitation amplitude was 10 mV, and the frequency range was 0.01 - 1000 kHz. Calculate the sodium ion diffusion coefficient D (cm 2 s -1 ) according to the formula.

[0043]

[0044] where R is the gas constant (8.314 J K -1 mol -1 ), T is the room temperature (298.15 K), A is the geometric surface area of the electrode (0.0314 cm 2 ), n is the number of electrons transferred (1), F is the Faraday constant (96485 C mol -1 ), C is the concentration of Na + (0.1 M), and σ is Z’ versus ω -1 / 2Slope of the plot (Z’ = Rs + Rct + σω -1 / 2 ).

[0045] 3 Experimental results

[0046] 3.1 Rheological properties of calcium-modified bacterial cellulose

[0047] Based on the biosynthesis of BC, calcium gluconate was used instead of glucose as the carbon source, and BCCa was produced by in-situ fermentation modification with FM883. The biosynthesis of BC is a complex reaction, including four key enzymatic steps, such as Figure 1 shown in a: (i) phosphorylation of glucose by glucokinase, (ii) isomerization of glucose-6-phosphate to glucose-1-phosphate by phosphoglucomutase, (iii) synthesis of uridine diphosphate glucose by pyrophosphorylase, and (iv) cellulose synthase reaction.

[0048] The obtained BC and BCCa are soft and can be restored after folding. Scanning electron micrographs show that nanofibers are randomly distributed in the radial-transverse plane in BC ( Figure 1 b). Fiber layers form a multi-layer structure on the cross-section of BCCa ( Figure 1 b), indicating the presence of certain crosslinking in BCCa, which may be due to the coordination of calcium ions expanding the pores. EDX spectra show that the calcium ion concentration in BCCa is increased compared to BC ( Figure 1 b), further confirming the successful incorporation of Ca 2+ .

[0049] The influence of BCCa on gel properties was further investigated by rheological behavior analysis. The viscosities of BC and BCCa decrease with increasing shear rate ( Figure 1 c), showing shear-thinning behavior. The maximum strain of BCCa increases ( Figure 1 d), indicating a decrease in its stiffness. As the temperature increases and decreases, the storage modulus (G') of BC and BCCa is greater than the loss modulus (G”) ( Figure 1 e,f), indicating that the hydrogel remains stable in terms of elasticity and still has the characteristics of a viscoelastic solid. The viscoelastic behavior is less affected by frequency changes ( Figure 1 g). The G′ and G″ of BCCa are lower than those of BC ( Figure 1 h), indicating a decrease in its resistance to strain. These data show that BCCa has both high stiffness and excellent elastic properties.

[0050] 3.2 Structural characterization of calcium-modified bacterial cellulose

[0051] BC and BCCa have typical cellulose Iα monoclinic diffraction patterns ( Figure 2a), the diffraction peaks are located at (100), (010), and (110). The XRD pattern is consistent with the standard curve of cellulose Iα ( Figure 2 b), indicating that the overall structures of BC and BCCa are Iα, but the crystallinity decreases from 36.77% (BC) to 26.65% (BCCa). This is because the introduction of calcium ions disrupts the intramolecular hydrogen bonds, resulting in the dissociation of some cellulose molecular chains, thus affecting the cellulose crystal structure.

[0052] The FT-IR spectra of BC and BCCa are similar. As Figure 2 shown in c, compared with BC, the OH stretching vibration absorption peak of BCCa shifts from 3349 cm -1 to 3405 cm -1 , the -CH- stretching vibration absorption peak shifts from 2897 cm -1 to 2922 cm -1 , and the stretching vibration absorption peak of -C=O shifts from 1664 cm -1 to 1656 cm -1 , indicating that Ca 2+ enters the BC network, thus affecting the -COOH and the intermolecular hydrogen bonds between cellulose chains. Ca 2+ coordinates with the cellulose chains, thus weakening the hydrogen bond strength between cellulose chains; on the other hand, Ca 2+ can reduce the repulsive force between cellulose chains.

[0053] The XPS spectra of BCCa show peaks at 286.56, 347.44, and 532.91 eV, which are attributed to the core levels of C1s, Ca2p, and O1s, respectively ( Figure 2 d, e). In the C1s spectrum of BCCa ( Figure 2 d), the binding energy peak is decomposed into 4 spectral bands, namely 289.26, 288.05, 286.56, and 284.8 eV, which can be attributed to COO, C=O, C-O / C-N, and C-C / C-H, respectively. In the Ca2p spectrum of BCCa ( Figure 2 e), the binding energy peak is located at 347.44 eV, which can be attributed to Ca 2+ . According to the research on different calcium ion energy levels by Demri et al. (1995), it is inferred that there is a CO-Ca bond in BCCa. The solid-state 13 13C NMR spectrum is as Figure 2 shown in f. In BC, the acidic form of carboxyl appears at 171 ppm, while in BCCa, the Ca 2+ -bound form shifts to 175 ppm, and this shift is attributed to the coordination of Ca 2+ ions with carboxyl groups.

[0054] Figure 2Figure e shows the TG and DTG curves of BCCa. During the process from room temperature to 200 °C, the weight loss is 7.18%, which is caused by the evaporation of surface water; the mass loss from 200 to 800 °C is related to the depolymerization and decomposition of cellulose; the highest decomposition temperature of BCCa is 339.5 °C, which is higher than the highest decomposition temperature of BC (286.7 °C), indicating that the thermal stability of BCCa is increased due to the increased calcium coordination in BCCa; the DTG of BCCa is 10.18% / min, which is lower than 10.26% / min of BC. The TG and DTG analysis results show that the thermal stability of BCCa is higher than that of BC.

[0055] The sodium ion diffusion coefficient of BC obtained by electrochemical impedance spectroscopy is (8.51 ± 1.89)×10 -14 cm 2

[0056] s -1 , and the sodium ion diffusion coefficient of BCCa is (2.39 ± 0.98)×10 -12 cm 2 s -1 . It shows that the BCCa / Au electrode exhibits more favorable sodium ion transfer kinetics than the BC / Au electrode, further proving that BCCa enhances the diffusion of sodium ions.

[0057] Example 2

[0058] 1 Preparation of surimi gel

[0059] Take out the frozen Nemipterus surimi from the -20 °C freezer and thaw it at 4 °C until the central temperature reaches 0 °C. Put a certain amount of surimi (85%) and an appropriate amount of ice into a meat grinder (Braun FP3010), and stir the mixture at a speed of 200 - 300 rpm for 10 min until the surimi becomes viscous. After adding 2% or 1.5% NaCl, stir at 600 rpm for 2 min, then add 0.5% BC or 0.5% BCCa and continue to stir for 1 min. Manually extrude the pellets into shape (about 20 g each). Keep in a constant temperature water bath at 40 °C for 60 min and at 90 °C for 20 min to form surimi gel, and immediately ice bath for 30 min to obtain fish balls. The samples containing 1.5% NaCl and bacterial cellulose are labeled as CK1.5% + BC0.5% and CK1.5% + BCCa0.5% respectively. The samples containing 1.5% NaCl and 2.0% NaCl are labeled as CK1.5% and CK2% respectively.

[0060] 2 Analysis methods for surimi gel properties

[0061] 2.1 Effect of BCCa on sodium ion diffusion rate and sodium ion release analysis

[0062] The surimi gel balls were prepared in water at 40 °C and 90 °C respectively, and the Na + concentration was measured using a DWS-51 sodium ion analyzer (Shanghai Yidian Scientific Instrument Co., Ltd.). The Na + diffusion rate was calculated as follows:

[0063] Na + Diffusion rate (%) = (Na + concentration × volume of deionized water used in the water bath) / (mass of surimi gel before the water bath × NaCl addition ratio) Formula (1)

[0064] Sodium ion release was measured using a DWS-51 sodium ion meter (INESA Scientific Instrument Co., Ltd., China) and the method described by Zhang et al. (P. Zhang et al., 2024). The prepared surimi gel balls were cut into 8 pieces and pulverized for 15 seconds using a JR69-B175 meat grinder (Supco Co., Ltd., Zhejiang, China). 10 g of the pulverized surimi gel was placed in a 6×8 cm nylon bag and soaked in 200 mL of deionized water (containing 0.5 mL of concentrated ammonia water), with continuous magnetic stirring. The sodium ion release value was recorded every 5 seconds within 5 minutes.

[0065] 2.2 Effect of BCCa on taste

[0066] 2.2.1 Sensory evaluation

[0067] According to the method of Huang et al. (Huang et al., 2024), with informed consent, 9 food science majors with sensory evaluation experience and no taste or olfactory disorders were selected to participate in this experiment. During the sensory evaluation, each evaluator randomly tasted the sample for 10 s, then spat it out and recorded the corresponding sensory indexes. Before the next evaluation, an interval of 2 min was required, and the mouth was rinsed with pure water. The evaluation criteria are shown in Table 1.

[0068] Table 1 Sensory evaluation criteria

[0069]

[0070]

[0071] 2.2.2 Electronic tongue detection

[0072] According to the method described by Huang et al. (Huang et al., 2024), the flavor characteristics of surimi gels were analyzed using an electronic tongue (INSENT SA402B, Japan). The collection time was set to 120 s, and the detection time for each sample was 30 s. During the analysis, 15 g of the ground surimi gel was added to 150 mL of deionized water at 37 °C and magnetically stirred for 1 min. After vacuum filtration of the extract, the filtrate was centrifuged at 4000 rpm / min for 10 min to obtain the supernatant for determination.

[0073] 2.3 Influence of BCCa on flavor

[0074] The method of Xu et al. (Xu et al., 2020) was adopted to analyze the influence of BCCa on flavor using an electronic nose (AIRSENSE Pen3, Germany) equipped with 10 sensor arrays. Each sensor is sensitive to hydrogen sulfide, sulfides, alcohols, ketones, aldehydes, and aromatic compounds. After the treatment described in the sample preparation section, 2 g of the surimi gel sample was stored in a sealed 25 mL glass gas collection bottle, water-bathed at 50 °C for 20 min, and then subjected to electronic nose analysis. 20 mL of the headspace was extracted with a needle and injected into the sensor array chamber through a sampling tube. The cleaning stage included pumping clean air for 120 s to normalize the sensor signals between samples. The sampling pump ran at a constant speed of 0.4 L / min for 180 s. The response value of the sensor per second was recorded. Each sample was repeated three times.

[0075] 2.4 BCCa on cooking loss measurement, whiteness, water content, and water holding capacity of BCCa

[0076] Cooking loss determination: Weigh approximately 12 g of raw surimi at room temperature, extrude it into pellets, and record the weight before cooking (W1). After a 1 h water bath at 40 °C and a 20 min water bath at 90 °C, immediately cool it, wipe off the surface moisture with absorbent paper, and record the weight after cooking (W2). The formula for calculating cooking loss is:

[0077] Cooking loss (%) = (W1 - W2) / W2 × 100% Formula (2)

[0078] Whiteness determination: Cut a 1.5 cm surimi gel and immediately measure the center part using a color difference meter. After calibrating with a standard plate, the brightness value (L * ), red value (a * ), and yellow value (b * ) were measured through a full-automatic color difference meter. The formula for calculating the whiteness value is:

[0079] Whiteness value = 100 - [(100 - L * ) 2 + (a * ) 2+(b * ) 2 0.5 Formula (3)

[0080] Determination of moisture content: The moisture content (WC) was determined by the drying method (Xia et al., 2025). The raw materials were ground with a small blender, 2.0 g of each sample was weighed, and dried in an oven at 105 °C until the weight change was less than 2 mg. The weight change of the sample was recorded to calculate WC.

[0081] Determination of water holding capacity: A certain amount of fish balls (M1) were wrapped with absorbent paper and placed in a centrifuge tube, centrifuged at 4 °C and 3000 r / min for 10 min, and the mass after centrifugation (M2) was recorded. The formula for calculating the water holding capacity (WHC) is:

[0082] Water holding capacity (%) = (M2 / M1) × 100% Formula (4)

[0083] 2.5 Effects of BCCa on gel strength and texture

[0084] The gel strength of surimi gel was evaluated using a TMS-Touch texture analyzer (FTC, USA) according to the method of Zhang et al. (P. Zhang et al., 2024). The surimi gel was cut into 1×1×1 cm for testing. A 12.7 mm probe was used, the trigger force was 0.75 N, and the puncture distance was 6 mm. The prediction and test speeds were both set at 60 mm / min.

[0085] According to the protocol described by Zhang et al. (P. Zhang et al., 2024), TPA analysis of surimi gel was performed using a TMS-Touch texture analyzer (FTC, USA). The surimi gel balls were cut into 1×1×1 cm for testing. A 12.7 mm probe was used, the trigger force was 0.5 N, and the deformation was 40%. The test speed was maintained at 60 mm / min.

[0086] 2.6 Water distribution in surimi gel

[0087] A low-field nuclear magnetic resonance analyzer (Meso MR20-0660H-I, Jiangsu, China) was used to measure the water distribution and nuclear magnetic resonance imaging (MRI) according to the method of Zhang et al. (S. Zhang et al., 2024). The measurement was performed on the whole surimi gel ball. The CONTIN algorithm was used to process the exponential decay graph to obtain the T2 relaxation curve and peak. MRI imaging of the surimi gel sample was performed to obtain the proton density image, and the water distribution of the sample was analyzed using a pseudocolor map.

[0088] 2.7 Effects of BCCa on the structural properties of low-salt surimi gel ​

[0089] FTIR was performed by the KBr pellet method, with a wavelength range of 400 to 4000 cm -1 , and the sampling interval was 0.5 cm -1 (USA ThermoFisher Scientific Nicolet iS20). The XRD pattern was scanned between 10° and 50° (2θ) with a step interval of 0.02° (Empyrean Rigaku SmartLab SE, Japan).

[0090] 3 Experimental results

[0091] 3.1 Effect of BCCa on the Na + diffusion rate and Na + release analysis

[0092] The total Na + diffusion rate in surimi gels in a 40 °C and 90 °C water bath was as Figure 3 shown in a, ranging from 26.71 ± 0.09% to 29.04 ± 0.21%. After reducing the salt content, the Na + diffusion rate in surimi gels decreased. Adding bacterial cellulose could increase the Na + diffusion rate in surimi gels, and the Na + diffusion rate in the surimi gel treated with BCCa was the highest, reaching 29.04 ± 0.21%. The results showed that BCCa could significantly promote the Na + diffusion.

[0093] The Na + release from surimi gels was as Figure 3 shown in b. After reducing the salt content, the Na + release from surimi gels decreased. Adding BCCa could accelerate the Na + release from surimi gels, and the final release amount was higher than that of CK by 1.5% (low-salt group). The Na + release within 20 s was as Figure 3 shown in c. The Na + release amount of CK1.5% + BCCa0.5% was significantly higher than that of CK1.5%, and was comparable to that of CK2%. It indicated that BCCa could significantly promote the Na + release.

[0094] The overall flavor characteristics of the samples were further analyzed using an electronic tongue and sensory evaluation ( Figure 3 d, Table 2). The results of the electronic tongue showed that ( Figure 3d), The flavorless thresholds of the reference solution containing 30 mM KCl and 0.3 mM tartaric acid were: sourness -13, saltiness -6, and other indicators 0 (He et al., 2024). For all surimi gel samples, the B-values of sourness, astringency, and aftertaste were lower than the flavorless thresholds. Adding BCCa did not have an adverse effect on bitterness, aftertaste-A, umami, richness, or saltiness in the CK1.5% group. Figure 3 e, f showed that BCCa enhanced the saltiness and umami of surimi (p<0.05). The saltiness score of CK1.5%+BCCa0.5% was -1.26( Figure 3 e), which was 30% higher than that of CK1.5% (-1.80) because BCCa promoted the diffusion rate and release of Na + in the surimi gel containing NaCl, thus enhancing the saltiness perception of the surimi gel. In addition, the umami score of CK1.5%+BCCa0.5% was 0.88( Figure 3 f), which was significantly higher than that of the low-salt group (CK1.5%) and 1.96 times that of the high-salt group (CK2%). Previous studies have shown that umami substances can enhance people's perception of saltiness, not only reducing salt intake but also improving flavor.

[0095] The effects of BCCa on the sensory evaluation of surimi gel are shown in Table 2. First, the saltiness score of CK1.5%+BCCa0.5% was significantly higher than that of CK1.5% (P<0.05) and was comparable to that of CK2%. Second, BCCa increased the scores of structure, hardness, elasticity, and color, making them closer to the CK2% high-salt group. The overall evaluation score of CK1.5%+BCCa0.5% was higher than that of CK1.5% (P<0.05). The weight ratios of each index were: taste (saltiness) 0.4, tissue state 0.1, hardness 0.1, elasticity 0.2, color 0.1, and overall evaluation 0.1. According to the weight ratios of each index, the weighted scores were obtained, and CK1.5%+BCCa0.5% was significantly higher than CK1.5% (P<0.05) and was comparable to the high-salt group (CK2%). In summary, BCCa improved the saltiness perception and overall quality of low-salt surimi.

[0096] Table 2 Sensory evaluation of surimi gel

[0097]

[0098] Note: Different lowercase letters in the table indicate significant differences at the 0.05 level detected by the Duncan method.

[0099] 3.2 Effects of BCCa on appearance, flavor, whiteness, cooking loss, moisture content, water holding capacity, gel strength, and texture characteristics

[0100] No obvious differences were found in the appearance and internal structure of fish balls added with BC and BCCa( Figure 4 a). The electronic nose radar chart shows that the sensor response profiles of all samples are similar( Figure 4 b), indicating the similarity in the distribution of volatile organic compounds (VOCs). The signal values of S7 (sensitive to sulfides and terpenes) and S8 (sensitive to alcohols and aldehyde-ketone compounds) in CK1.5% + BCCa0.5% are higher than those of other sensors (p<0.05), which indicates that BCCa affects the VOCs of surimi gel.

[0101] The whiteness of fish balls added with BC and BCCa is as Figure 4 shown in c. Compared with the high-salt group (CK2%), the whiteness of surimi gel decreases after salt reduction (CK1.5%). This is because the content of salt-soluble proteins decreases after salt reduction, resulting in a decrease in the whiteness value of surimi gel. The whiteness increases after adding BCCa. The higher the whiteness, the higher the quality of surimi and its products, and the more easily it is accepted by consumers.

[0102] The cooking loss of fish balls added with BC and BCCa is as Figure 4 shown in d. Compared with CK2%, the cooking loss of CK1.5% increases from 6.54±0.26% (CK2%) to 10.19±0.17% (CK1.5%) after salt reduction. The cooking loss decreases after adding BCCa, and it is 5.06±1.05% for CK1.5% + BCCa0.5%.

[0103] The WC and WHC of fish balls added with BC and BCCa are as Figure 4 shown in e and Figure 4 f. Compared with the high-salt group (CK2%), the WC decreases from 20.29±0.10% (CK2%) to 19.63±0.10% (CK1.5%), and the WHC decreases from 94.54±0.24% (CK2%) to 91.75±0.53% (CK1.5%) after salt reduction. After adding BC and BCCa, both WC and WHC increase, and the WC and WHC of CK1.5% + BCCa0.5% increase to 20.18±0.04% and 95.08±0.47% respectively. On the one hand, BCCa improves the compactness and orderliness of surimi gel, enhancing the moisture content and water holding capacity. On the other hand, BCCa itself has water holding capacity, which helps to retain water in the system.

[0104] The effects of BCCa on the gel strength and texture of fish balls are as Figure 4As shown in g and h. Gel strength and texture are important parameters of fish ball gel and affect the quality of surimi gel. Compared with CK2%, the gel strength and hardness of fish balls after salt reduction (CK1.5%) decreased. The gel strength, hardness and adhesiveness of fish ball gel added with BCCa increased significantly (p < 0.05) (Table 3). This is due to the filling effect of BCCa and the enhancement of the surface charge of cellulose on fish ball gel.

[0105] Table 3 Texture of fish ball gel

[0106]

[0107] Note: Different lowercase letters in the table indicate significant differences at the 0.05 level detected by Duncan's method.

[0108] 3.3 Effect of BCCa on water distribution

[0109] Salt affects the distribution and migration of water during surimi processing. As Figure 5 shown in a, LF-NMR analysis showed that after multi-exponential fitting, three peaks appeared: T 2b , T 21 and T 22 , representing bound water, non-fixed water and free water respectively. MRI images ( Figure 5 b) showed that the water content of surimi decreased after salt reduction, while the water content of surimi increased significantly after adding BC and BCCa. In the CK1.5% + BCCa 0.5% group, the red area in the MRI image increased and the hydrogen proton density rose, indicating that the network captured more water and achieved a more uniform water distribution, improving the water retention and texture stability of fish balls. Consistent with Figure 4 the results, BCCa enhanced the gel properties of surimi. Further analysis found that after salt reduction, both the bound water and immobilized water in fish balls decreased (the A 21 and P 21 values decreased), while BCCa could increase both the bound water and free water and decrease the immobilized water in low-salt fish balls ( Figure 5 c, d). This phenomenon may be attributed to the fact that the cellulose network can enhance the water retention of bound water and capture more free water.

[0110] 3.4 Effect of BCCa on the structural properties of low-salt surimi gel

[0111] Figure 6 a is the FT-IR diagram of fish balls. In CK2%, 3410 cm -1 (amide A band, O-H / N-H stretching vibration), 2924 cm -1 (amide B band, N-H / =C-H stretching vibration), 1653 cm -1(Amide I band, C=O / C=N stretching vibration), 1545 cm -1 (Amide II band, N-H bending and C=N stretching vibration) and 1054 cm -1 (C-O and C-C stretching vibrations), etc. absorption peaks (He etal., 2024). After adding BC, the characteristic peaks of amide I and amide II did not change significantly, indicating that BC mainly affects the hydrogen bond structure between proteins and water molecules. In CK1.5% + BCCa0.5%, after introducing Ca 2+ , the absorption peak of the amide I band shifted from 1653 cm -1 to 1659 cm -1 , indicating that there was an interaction between the amino nitrogen atom, carboxyl oxygen atom, and carbonyl oxygen atom of the peptide segment and Ca 2+ , affecting the secondary structure of the protein.

[0112] Figure 6 The secondary structure results shown in Fig. b indicate that CK1.5% + BCCa0.5% promoted the conformational transition from β-turns and random coils to α-helices and β-sheets, indicating that the internal hydrogen bond network became denser, resulting in a more ordered and stable gel structure.

[0113] XRD patterns ( Figure 6 Fig. c) revealed the effects of different treatment methods on the crystal structure of the samples. All samples showed broad diffraction peaks near 2θ = 20°, and sharp and intense peaks at 2θ = 29.5° and 31.8°, indicating the presence of amorphous regions and crystal structures with similar crystal characteristics. No new peaks were observed in the samples added with BC and BCCa, indicating that no new crystal structures were formed. However, the changes in peak intensity indicated that the intermolecular interactions had changed. The crystallinities of CK2%, CK1.5%, CK1.5% + BC0.5%, and CK1.5% + BCCa0.5% were 65.21 ± 1.02%, 39.44 ± 0.20%, 87.28 ± 5.15%, and 97.35 ± 7.71%, respectively, indicating that the structure of CK1.5% + BCCa0.5% was more ordered and denser.

[0114] 3.5 Microstructure of surimi gel and NaCl distribution

[0115] The microstructure of surimi gel is as shown in Figure 7As shown in a), there are obvious differences in the microstructures of different samples. The surface structures of CK2% and CK1.5% surimi gels are rough. BC and BCCa improve the microstructure of surimi gels. The microstructure of CK1.5% + BCCa0.5% surimi gel is more continuous, uniform, orderly, and dense than that of the control group CK1.5%. The hydrogen bond and filling effects of BCCa make the microstructure of surimi gels dense and orderly. The dense pore structure can improve the texture, accelerate the diffusion and release of sodium ions, and enhance the perception of saltiness. The EDS results ( Figure 7 b) show that the concentrations of Cl and Na in CK1.5% are significantly lower than those in CK2%, decreasing from 0.301 ± 0.040% to 0.80 ± 0.04% and from 1.757 ± 0.052% to 1.45 ± 0.04% respectively. After adding BCCa, the Na concentration in CK1.5% + BCCa0.5% increases from 1.45 ± 0.04% to 1.539 ± 0.042%. The results indicate that BCCa can affect the ion distribution by changing the microstructure.

[0116] In summary, BCCa can significantly enhance the saltiness perception and texture of low-salt surimi gels. On the one hand, the calcium ion coordination expands the spacing between BC molecular chains, which is beneficial to the entry and transportation of Na + . On the other hand, BCCa makes the structure of surimi gels more orderly and dense through hydrogen bond interactions, and can transform the secondary structure of the gel into a more stable gel structure. This improvement enhances the diffusion and release of Na + in low-salt surimi gels, gel strength, hardness, microstructure, and sensory properties. These research results show that BCCa can be used as a valuable additive in the processing industry of aquatic and livestock meat products. On the premise of maintaining the flavor and texture of aquatic and livestock meat products, it can significantly reduce the salt addition in aquatic and livestock meat products and enhance the saltiness perception, meeting the current trend of consumers' preference for a healthier diet.

[0117] It should be noted that although the above embodiments have been described in this article, the patent protection scope of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, any changes and modifications made to the embodiments described in this article, or equivalent structural or equivalent process transformations made using the content of the specification and drawings of the present invention, and the direct or indirect application of the above technical solutions to other related technical fields are all included in the patent protection scope of the present invention.

Claims

1. A calcium-modified bacterial cellulose, characterized in that, The calcium-modified bacterial cellulose described above is obtained by in-situ fermentation of Gluconacetobacter xylinus in a liquid medium using calcium gluconate as a carbon source through an ion coordination method.

2. The calcium-modified bacterial cellulose according to claim 1, wherein The components of the liquid medium include 40 - 50 g / L of calcium gluconate, 0.8 - 1.2 g / L of KH2PO4, 4 - 6 g / L of yeast extract, 10 - 20 g / L of MgSO4, 4 - 6 g / L of peptone, 0.8 - 1.2 g / L of citric acid, and 1.5 - 2.5% ethanol (volume percentage).

3. The calcium-modified bacterial cellulose according to claim 1 or 2, characterized in that, The calcium-modified bacterial cellulose has an alpha crystal structure with a crystallinity of 26 - 27%.

4. A method for preparing calcium-modified bacterial cellulose as described in any one of claims 1-3, characterized in that, The preparation method described is to add the seed solution of Gluconacetobacter xylinus to the liquid medium at an addition amount of 6-10% by mass percentage, and incubate the culture at 25-35 °C for 6-8 days; soak the obtained membrane vesicles in an edible alcohol solution with a volume percentage of 70-80% for 2-3 days, and then boil in pure water for 1-2 h; the bacterial concentration of the seed solution is (3.0-4.0)×10 8 cfu / mL.

5. A low-salt livestock gel, characterized in that, The gel contains 80 - 90% of aquatic animal meat mince, 0.5 - 1% of calcium-modified bacterial cellulose, 1.5 - 2% of NaCl, and the rest is water, all of which are mass percentages.

6. The low-salt aquaculture gel according to claim 5, characterized in that, The preparation method of the gel is as follows: thaw the frozen aquatic animal meat mince at 2 - 8 °C until the central temperature reaches 0 - 4 °C. Put 80 - 90% of the aquatic animal meat mince and ice into a meat grinder and stir the mixture at a speed of 200 - 400 rpm for 8 - 12 min until the aquatic animal meat mince becomes viscous. After adding 1.5 - 2% of NaCl, stir at 500 - 700 rpm for 2 - 3 min, then add 0.5 - 1% of calcium-modified bacterial cellulose and stir for 1 - 2 min, and form pellets by hand. Keep it in a constant temperature water bath at 35 - 45 °C for 50 - 70 min and then at 85 - 95 °C for 15 - 25 min to form an aquatic animal meat mince gel. Immediately cool it in an ice bath for 25 - 35 min to obtain the gel, and all the percentages above are mass percentages.

7. Use of the calcium-modified bacterial cellulose according to any one of claims 1 - 4 in low-salt aquatic animal meat mince products.

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