Preparation method and application of microbial composite membrane for improving flavor and quality of cured meat

Abstract

The invention discloses a preparation method and application of a microbial composite membrane for improving flavor and quality of cured meat, and belongs to the technical field of meat curing. The microbial composite membrane is composed of an outer bacterium-blocking breathable layer and an inner functional bacterium loading layer, the outer layer is an electrostatic spinning bacterium-blocking breathable layer with the pore diameter being 0.1-0.5 mu m, and the inner layer is a functional bacterium loading layer with the pore diameter being 0.1-0.5 mu m; harmful microorganisms larger than or equal to 99.99% can be intercepted, the air permeability is larger than or equal to 5 mm / s (100 Pa), and normal proceeding of fat oxidation and beneficial microorganism metabolism is guaranteed; the inner layer is a freeze-drying functional bacterium loading layer fixed by a water-soluble polymer, the microorganism loading density is 10-1000CFU / cm < 2 >, and the freeze-drying functional bacterium loading layer can quickly recover and play a ripening role after contacting with the surface of meat or absorbing moisture, so that directional microorganism regulation and control in the meat ripening process are realized, and the ripening efficiency and quality are improved. The microbial composite membrane for improving the flavor and quality of the cured meat has triple functions of bacterium blocking, ventilation and functional bacterium loading, and is suitable for replacing a traditional natural fermentation or exposed curing mode. Through the synergistic effect of bacterium blocking, ventilation and directional inoculation, traditional natural fermentation is replaced, the curing time is shortened by more than or equal to 20%, the infectious microbe pollution risk is reduced, and the meat curing efficiency and safety are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of meat ripening, and particularly relates to a preparation method and application of a microbial composite film for improving the flavor and quality of ripened meat. BACKGROUND

[0002] Meat ripening is a meat processing method with a long history and wide application, and plays an important role in the production of traditional hams and matured steaks. The core role of meat ripening is to improve the tenderness, flavor and overall quality of meat. The ripening process relies on the synergistic action of microbial metabolism, endogenous enzymolysis and environmental factors such as temperature, humidity and gas composition, so that the proteins and fats in meat are gradually degraded to generate free amino acids and volatile flavor substances, thereby giving meat a unique ripening flavor. Traditional meat ripening requires low temperature (0-4℃), high humidity (75%-85%) and a controllable ventilation environment for several weeks to several months, and a microbial bacterial film is formed on the surface to give a special flavor. The ripening process of Spanish Iberian ham is a traditional and delicate fermentation-drying process, which takes as long as 18-36 months. However, "bare ripening" directly exposes meat pieces to air, and the following long-standing problems have not been solved:

[0003] 1. High risk of mixed bacteria - Listeria, Salmonella, Penicillium, Aspergillus and other bacteria will cause the whole piece to be scrapped once they colonize, resulting in significant economic losses;

[0004] 2. Long cycle - several weeks to several months, low efficiency;

[0005] 3. High salt dependence - excessive salting to inhibit bacteria, which goes against the trend of low-sodium diet;

[0006] 4. Large quality fluctuation - uncontrollable natural flora, significant fluctuation in flavor and quality.

[0007] To reduce the risk, wet ripening (such as vacuum-packed steaks) is proposed, which uses self-enzymolysis to improve tenderness and reduce some of the risks of mixed bacteria at 0-4℃. However, due to the lack of oxygen, the reproduction of beneficial microorganisms on the surface of meat is limited, and wet ripening is difficult to significantly improve the flavor, and its application effect is limited.

[0008] With the progress of material science, nanofibers are widely used in food packaging research due to their high specific surface area and controllable pore structure. By electrospinning, natural antibacterial agents such as nisin, tea polyphenols and chitosan, or inorganic antibacterial agents such as nano-silver and zinc oxide, can be loaded into nanofibers to prepare food preservative films with antibacterial release function. This kind of packaging can effectively inhibit the growth of spoilage bacteria such as Escherichia coli and Staphylococcus aureus, and prolong the preservation period of meat due to its good barrier property. However, these nanofiber-based food preservation materials aim to "comprehensively inhibit bacteria", and do not distinguish between beneficial ripening bacteria and harmful miscellaneous bacteria, so they are not suitable for meat ripening processes that rely on specific beneficial microorganisms.

[0009] Therefore, there is an urgent need to develop a new type of intelligent packaging material that can effectively block the invasion of harmful microorganisms from the outside, and at the same time, can release or load beneficial ripening bacteria on the surface of meat in a targeted manner, and maintain a suitable gas and moisture permeable microenvironment to support their metabolic activities, thereby reproducing or even enhancing the flavor and texture of traditional ripening under the premise of safety, providing a new technical path for meat ripening that breaks through the limitations of traditional processes. SUMMARY

[0010] To solve the above technical problems, the present application provides a preparation method and application of a microorganism composite film for improving the flavor and quality of ripened meat. The microorganism composite film for improving the flavor and quality of ripened meat of the present application loads microorganisms beneficial to meat ripening, has the functions of synergistic antibacterial and gas permeability, and the double-layer design and targeted microorganism regulation of the present application provide a new technical path for meat ripening, which has application value.

[0011] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0012] The present application provides a microorganism composite film for improving the flavor and quality of ripened meat, which comprises an antibacterial and gas permeable layer and a functional bacteria loading layer, and the functional bacteria loading layer is located inside the antibacterial and gas permeable layer (i.e. the functional bacteria loading layer is used as the side in contact with meat).

[0013] The antibacterial and gas permeable layer is used to block the entry of external microorganisms, prevent harmful bacteria or harmful fungi from reproducing on the surface of meat, thereby avoiding meat spoilage, and at the same time has excellent gas permeability, which does not affect the oxidation of fat and the aerobic metabolism of beneficial microorganisms during the ripening process.

[0014] The functional bacteria loading layer loads freeze-dried microorganisms beneficial to meat ripening, which releases microorganisms after contacting the surface of meat or absorbing moisture in the environment, and promotes meat ripening.

[0015] The application first proposes a microbial composite film for improving the flavor and quality of cooked meat, which is a double-layer multifunctional composite nanofiber film specially used for meat cooking, and is composed of an outer layer of bacteria-resistant and air-permeable layer and an inner layer of functional bacteria loading layer; the outer layer of bacteria-resistant and air-permeable layer is prepared by electrospinning, has controllable pore size and good air permeability, can efficiently intercept ≥99.99% harmful miscellaneous bacteria, and at the same time maintain appropriate oxygen transmission to ensure normal fat oxidation and beneficial bacteria metabolism; the inner layer of functional bacteria loading layer introduces freeze-dried functional bacteria, which are fixed by water-soluble polymers and can quickly recover and dominate the cooking process after absorbing moisture or contacting the surface of meat. Unlike the existing "single-layer bacteria-resistant film" or "overall bacteria-inhibiting packaging", the double-layer cooperative structure of the application not only ensures the purity and stability of the cooking environment, but also actively regulates the establishment of beneficial bacteria, and there is currently no such "bacteria-resistant + air-permeable + directional inoculation" integrated design. After applying the microbial composite film of the application, the meat cooking period can be shortened by ≥20%, the meat weight loss rate is reduced, and the flavor consistency is significantly improved, which means that on the industrialization level, high-quality, low-salt and safe cooked meat products can be produced while reducing risks and costs.

[0016] Further, the loading density of the freeze-dried microorganisms in the functional bacteria loading layer is 10-1000 CFU / cm 2 .

[0017] Further, the freeze-dried microorganisms are selected from freeze-dried fungi, freeze-dried fungal spores or freeze-dried bacteria.

[0018] Further, the freeze-dried microorganisms are selected from freeze-dried fungi, freeze-dried fungal spores or freeze-dried bacteria.

[0019] Further, the pore size of the bacteria-resistant and air-permeable layer is 0.1-0.8 μm, the thickness is 0.3-10 μm, the air permeability is not less than 5 mm / s when the test pressure difference is 100 Pa, and the interception efficiency for Bacillus atrophaeus spores is not less than 99.99% when the test flow rate is 2.8 L / min for 15 minutes.

[0020] Further, the raw material for preparing the bacteria-resistant and air-permeable layer is a non-water-soluble polymer.

[0021] Further, the non-water-soluble polymer is selected from at least one of polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polystyrene (PS), polyether sulfone (PES), polyvinylidene fluoride (PVDF), polyimide (PI), corn protein zein (Zein) and polyacrylonitrile (PAN).

[0022] Further, the functional bacteria in the functional bacteria loading layer are fixed by a water-soluble polymer.

[0023] Further, the water-soluble polymer is selected from at least one of polyvinyl alcohol (PVA), polyethylene oxide (PEO), vinylpyrrolidone (PVP), sodium alginate (SA), carboxymethyl cellulose, corn protein, starch, guar gum and gum arabic.

[0024] The application also provides a preparation method of the microbial composite film for improving the flavor and quality of cooked meat.

[0025] After mixing the microbial solution and the water-soluble polymer solution, the microbial solution is loaded on the inside of the bacteria-resistant and air-permeable layer by spraying or electrospinning, and then freeze-drying treatment is performed to obtain the microbial composite film for improving the flavor and quality of cooked meat.

[0026] Alternatively, the microbial solution is first sprayed on the inside of the bacteria-resistant and air-permeable layer, then the microbial solution is fixed by spraying or electrospinning with a water-soluble polymer solution, and finally freeze-drying is performed to obtain the microbial composite film for improving the flavor and quality of cooked meat.

[0027] Alternatively, the microbial solution is pre-freeze-dried into powder, the powder is distributed on the inside of the bacteria-resistant and air-permeable layer by electrostatic spraying, and then fixed by spraying or electrospinning with a water-soluble polymer solution to obtain the microbial composite film for improving the flavor and quality of cooked meat.

[0028] Further, the preparation method of the microbial solution is as follows: dispersing the microorganisms in PBS buffer (pH = 7.0) to obtain a microbial solution with a viable bacterial concentration of ≥1×10 3 CFU / mL, preferably a viable bacterial concentration of ≥1×10 5 CFU / mL.

[0029] The application also provides the use of the microbial composite film for improving the flavor and quality of cooked meat in meat cooking, wherein the functional bacteria loading layer is in contact with the meat.

[0030] Compared with the prior art, the application has the following advantages and technical effects:

[0031] 1. The application first uses a nanofiber bacteria-resistant and air-permeable film for meat cooking, breaking through the tradition of "bare cooking" for more than 100 years, effectively blocking harmful microorganisms (interception efficiency ≥ 99.99%), while maintaining air permeability (≥5 mm / s, 100 Pa), supporting fat oxidation and beneficial microbial metabolism.

[0032] 2.The application is a unique double-layer synergistic structure design, the microbial composite film is composed of an outer layer of bacteria-resistant and air-permeable layer and an inner layer of functional bacteria-loaded layer, both of which work synergistically. The bacteria-resistant and air-permeable layer prevents the invasion of foreign bacteria while providing air permeability; the functional bacteria-loaded layer fixes the freeze-dried beneficial bacteria through water-soluble polymers, and quickly recovers after contacting the surface of meat or absorbing moisture, and directionally regulates the ripening process. The use of the bacteria-resistant and air-permeable layer alone will inhibit the effect of beneficial microorganisms, and the inoculation of beneficial bacteria alone will also be difficult to avoid the pollution of external miscellaneous bacteria. The double-layer design of the application makes the advantages of the two complementary, and works synergistically, ensuring the safety and flavor stability of ripening.

[0033] 3.The application is aimed at the problems of long ripening period (several weeks to several months), high salt dependence, unstable flavor and high risk of miscellaneous bacteria pollution, through the "bacteria-resistant-air-permeable-directional inoculation" mechanism, the ripening time is shortened by ≥20%, the weight loss rate is reduced, the flavor consistency is improved, and the dependence on high salt pickling is reduced, which meets the low-sodium diet trend. BRIEF DESCRIPTION OF DRAWINGS

[0034] The accompanying drawings, which form a part of this application, are used to provide a further understanding of the application, and the illustrative embodiments thereof, and are not intended for limiting the application. In the drawings:

[0035] Figure 1 A scanning electron microscope image of the prepared PLA bacteria-resistant and air-permeable layer;

[0036] Figure 2 A scanning electron microscope image of the prepared PCL bacteria-resistant and air-permeable layer;

[0037] Figure 3 A scanning electron microscope image of the prepared Zein bacteria-resistant and air-permeable layer. DETAILED DESCRIPTION

[0038] The various illustrative embodiments of the application will now be described in detail, which should not be considered limiting on the application, but rather as a description of certain aspects, features, and embodiments of the application.

[0039] It should be understood that the terms used in the present application are merely for describing particular embodiments, and are not intended to limit the application. In addition, for the numerical range in the present application, it is understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or stated range and any other stated value or intermediate value within the stated range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All literature and similar materials cited in this application, including but not limited to, patents, genetic code, scientific and / or technical articles, and / or treatises, are expressly incorporated by reference.

[0041] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is intended to include all such modifications and variations in the scope of the present application.

[0042] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0043] The embodiment of the present application provides a microbial composite film for improving the flavor and quality of cured meat, which comprises a bacteria-resistant and air-permeable layer and a functional bacteria loading layer inside the bacteria-resistant and air-permeable layer.

[0044] The bacteria-resistant and air-permeable layer is used to prevent external microorganisms from entering and prevent harmful bacteria or harmful fungi from breeding on the surface of meat, thereby avoiding meat spoilage, while having excellent air permeability and not affecting fat oxidation and aerobic metabolism of beneficial microorganisms during curing.

[0045] The functional bacteria loading layer is loaded with freeze-dried microorganisms beneficial to meat curing, which releases microorganisms after contacting the surface of meat or absorbing moisture in the environment, thereby promoting meat curing.

[0046] After the microbial composite film of the present application is applied, the curing time of meat can be shortened by more than 20%, the content of flavor substances is significantly higher than that of traditional naked curing at the same time, the sensory score is better, the weight loss rate is reduced by about 3-5%, and the stability and consistency of curing are greatly improved. The electrospinning and spraying process used in the present application is mature and reliable, has good universality and expandability, and provides a safe, efficient and controllable technical solution for meat fermentation and curing.

[0047] In the embodiment of the present application, the loading density of freeze-dried microorganisms in the functional bacteria loading layer is 10-1000 CFU / cm 2 .

[0048] In embodiments of the present application, the freeze-dried microorganism is selected from a freeze-dried fungus, a freeze-dried fungus spore, or a freeze-dried bacterium, which is capable of recovering and exerting a ripening effect after absorbing water or contacting the surface of meat. Illustratively, the microorganism is selected from at least one of a freeze-dried fungus or a freeze-dried fungus spore beneficial to meat ripening, including but not limited to: a fungus of the genus Thamnidium or a yeast; a freeze-dried bacterium beneficial to meat ripening, including but not limited to: a lactic acid bacterium, a lactobacillus, or a staphylococcus xylosus.

[0049] In embodiments of the present application, the pore size of the bacteria-resistant air-permeable layer is 0.1-0.8 μm, the thickness is 0.3-10 μm, and the air permeability is not less than 5 mm / s when the test pressure difference is 100 Pa; and the interception efficiency for Bacillus marinus spores is not less than 99.99% when the test flow rate is 2.8 L / min for 15 minutes.

[0050] In embodiments of the present application, the raw material for preparing the bacteria-resistant air-permeable layer is a non-water-soluble polymer. Illustratively, the non-water-soluble polymer is selected from at least one of polylactic acid (PLA), polycaprolactone (PCL), polyurethane (PU), polystyrene (PS), polyethersulfone (PES), polyvinylidene fluoride (PVDF), polyimide (PI), corn protein zein, and polyacrylonitrile (PAN).

[0051] In embodiments of the present application, the method for preparing the bacteria-resistant air-permeable layer is: selecting at least one of PLA, PCL, PU, PS, PES, PVDF, PI, Zein, and PAN, dissolving the non-water-soluble polymer in an organic solvent to obtain a solution with a mass fraction of 5-25%, and electrospinning the solution to form a film. The product obtained by electrospinning is the bacteria-resistant air-permeable layer.

[0052] In embodiments of the present application, when preparing the bacteria-resistant air-permeable layer, a needle-type or needle-free electrospinning machine is used for electrospinning, the spinning temperature is controlled to be 25±4°C, the relative humidity is controlled to be 30-70%, and the substrate winding speed is controlled to be 0.2-6 m / min.

[0053] In embodiments of the present application, when preparing the bacteria-resistant air-permeable layer, the organic solvent is selected from acetone, ethanol, N,N-dimethylformamide, or dichloromethane.

[0054] In embodiments of the present application, when preparing the bacteria-resistant air-permeable layer, after electrospinning, the obtained film material is vacuum dried at 60°C for 12 h to remove residual solvent, and then sterilized by ultraviolet ozone treatment for 30 min.

[0055] In the embodiments of the present application, the water-soluble polymer is used to fix the functional bacteria in the functional bacteria loading layer. For example, the water-soluble polymer is selected from at least one of polyvinyl alcohol (PVA), polyethylene oxide (PEO), vinylpyrrolidone (PVP), sodium alginate (SA), carboxymethyl cellulose, corn protein, starch, guar gum and gum arabic. The present application can promote the curing of meat by dissolving and releasing the water-soluble polymer after contacting with meat.

[0056] The embodiments of the present application also provide a preparation method of the above-mentioned microbial composite film for improving the flavor and quality of cured meat, comprising the following steps:

[0057] After mixing the microbial solution and the water-soluble polymer solution, the mixture is loaded on the inside of the bacteria-resistant and air-permeable layer by spraying or electrospinning, and then freeze-drying treatment is performed to obtain the microbial composite film for improving the flavor and quality of cured meat;

[0058] Alternatively, the microbial solution is first sprayed on the inside of the bacteria-resistant and air-permeable layer, and then the microbial solution is fixed by spraying or electrospinning with the water-soluble polymer solution, and finally freeze-drying is performed to obtain the microbial composite film for improving the flavor and quality of cured meat;

[0059] Alternatively, the microbial solution is pre-freeze-dried into powder, the powder is distributed on the inside of the bacteria-resistant and air-permeable layer by electrostatic spraying, and then fixed by spraying or electrospinning with the water-soluble polymer solution to obtain the microbial composite film for improving the flavor and quality of cured meat.

[0060] In the embodiments of the present application, the preparation method of the microbial solution is as follows: the microorganisms are dispersed in PBS buffer (pH = 7.0) to prepare a microbial solution with a viable bacterial concentration ≥ 1 × 10 3 CFU / mL, preferably a viable bacterial concentration ≥ 1 × 10 5 CFU / mL.

[0061] In the preferred embodiments of the present application, the preparation method of the microbial composite film for improving the flavor and quality of cured meat uses any one of the following schemes A to C:

[0062] Scheme A: spraying / spinning after mixing the microorganisms and the water-soluble polymer

[0063] ① Preparation of microbial solution: the target microorganisms (such as at least one of Thamnidium fungi, yeast, lactic acid bacteria, lactobacillus and Staphylococcus xylosus) are dispersed in PBS buffer (pH = 7.0) to prepare a microbial solution with a viable bacterial concentration ≥ 1 × 10 5 CFU / mL;

[0064] ②Preparation of the loading mixture: the microbial solution is mixed with 5-20 wt% of a water-soluble polymer (such as PVA, PEO, PVP, SA, etc.) solution at a volume ratio of 0.001-0.1, and stirred uniformly to obtain the loading mixture;

[0065] ③Application and film formation: the loading mixture is uniformly applied to the inner surface of the bacteria-resistant and air-permeable layer using an electrostatic spraying device or an electrostatic spinning device, and the spraying amount per square centimeter is controlled at 0.01-0.5 mL, or the final gram weight per square meter of electrostatic spinning is controlled at 0.1-2 g / m 2 , to form a functional bacteria loading layer on the inner surface of the bacteria-resistant and air-permeable layer, and the loading density of the microorganisms in the functional bacteria loading layer is 10-1000 CFU / cm 2 , to obtain a composite film;

[0066] ④Freeze-drying treatment: the prepared composite film is placed in a vacuum environment of -60°C and ≤15 Pa for freeze-drying treatment, so that the microorganisms are in a freeze-dried dormant state while retaining their activity, to obtain a microbial composite film for improving the flavor and quality of cooked meat.

[0067] Scheme B: loading microorganisms first and then fixing with water-soluble polymers

[0068] ①Preparation of the microbial solution: the target microorganisms are dispersed in PBS buffer (pH = 7.0) to prepare a microbial solution with a viable bacterial concentration of ≥1×10 3 CFU / mL;

[0069] ②Spraying of the microbial solution: the microbial solution is uniformly sprayed on the inner surface of the bacteria-resistant and air-permeable layer using an electrostatic spraying machine or a pneumatic spraying device, and the spraying amount per square centimeter is controlled at 0.01-0.5 mL, so that the loading density of the microorganisms in the functional bacteria loading layer is 10-1000 CFU / cm 2 , and then the microorganisms are uniformly attached to the surface of the substrate after standing and drying at room temperature for 3 h to form a microbial layer;

[0070] ③Polymer fixing treatment:

[0071] A water-soluble polymer (such as PVA, PEO, PVP, SA, etc.) solution with a concentration of 5-20 wt% is prepared;

[0072] The water-soluble polymer solution is uniformly applied to the surface of the microbial layer using electrostatic spraying or electrostatic spinning, and the spraying amount is controlled at 0.01-0.5 mL / cm 2 , or the spinning gram weight is 0.1-2 g / m 2 , to achieve effective coating and fixing, and obtain a composite film;

[0073] ④ Freeze-drying treatment: freeze-drying the composite film at-60℃, ≤15Pa, to make the functional bacteria loading layer stable and dormant, and obtain the microbial composite film for improving the flavor and quality of cooked meat.

[0074] Scheme C: first prepare active freeze-dried microbial powder, and then fix with water-soluble polymer

[0075] ①Preparation of active freeze-dried microbial powder: freeze-drying the target microorganism at-60℃, ≤15Pa vacuum environment, to obtain stable active freeze-dried microbial powder;

[0076] ②Powder spraying: uniformly distribute the active freeze-dried microbial powder to the inner surface of the bacteria-resistant and air-permeable layer by electrostatic spraying or pneumatic spraying, to ensure that the loading density of microorganisms in the functional bacteria loading layer is 10-1000 CFU / cm 2 ;

[0077] ③Water-soluble polymer coating and fixing:

[0078] Prepare a water-soluble polymer (such as PVA, PEO, PVP, SA, etc.) solution with a concentration of 5-20wt%;

[0079] Spray or electrostatically spray the active freeze-dried microbial powder with the water-soluble polymer solution, and control the spraying amount per square centimeter to be 0.01-0.5mL or the gram weight to be 0.1-2g / m 2 ;

[0080] ④Freeze-drying treatment: dry again at-60℃, ≤15Pa, to ensure structural stability and retain microbial activity, and obtain the microbial composite film for improving the flavor and quality of cooked meat.

[0081] The embodiments of the present application also provide the application of the above-mentioned microbial composite film for improving the flavor and quality of cooked meat in meat cooking, which is covered on the surface of meat, and the side of the functional bacteria loading layer contacts the meat.

[0082] The microbial composite film of the present application is composed of an outer bacteria-resistant and air-permeable layer and an inner functional bacteria loading layer. The outer layer is an electrospun bacteria-resistant and air-permeable layer with a pore size of 0.1-0.5μm, which can intercept ≥99.99% harmful microorganisms and has an air permeability of ≥5mm / s (100Pa), to ensure normal fat oxidation and beneficial microbial metabolism. The inner layer is a freeze-dried functional bacteria loading layer fixed with water-soluble polymer, and the microbial loading density is 10-1000 CFU / cm 2After contacting or absorbing moisture from the meat surface, it can quickly recover and play a role in promoting ripening, realizing directional microbial regulation of the meat ripening process, and improving ripening efficiency and quality. The microbial composite film for improving the flavor and quality of ripened meat has the functions of bacteria blocking, air permeability and functional bacteria loading, and is suitable for replacing traditional natural fermentation or naked ripening. The present application replaces traditional natural fermentation through the synergistic effect of "bacteria blocking-air permeability-directional inoculation", shortens the ripening time by more than 20%, reduces the risk of contamination by miscellaneous bacteria, and improves the ripening efficiency and safety of meat.

[0083] Unless otherwise specified, the room temperature in the present application is 25±2℃.

[0084] The raw materials and reagents used in the embodiments of the present application are commercially available. As an example, Thamnidium elegans spore suspension, yeast Debaryomyces hansenii, Staphylococcus xylosus and Lactobacillus sakei are purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0085] Total volatile basic nitrogen is usually used to evaluate the freshness of meat, fish and other foods, because the decomposition of protein will produce volatile substances such as ammonia and amines, and the higher the TVB-N value, the more serious the corruption.

[0086] It should be pointed out that the details not described in the present application are conventional operating means in the art, and are not the focus of the present application, for example, the resuspension method of spore suspension, the preparation method of 10wt% polyvinyl alcohol aqueous solution, the preparation method of 5-20wt% water-soluble polymer solution, etc. The specific method is completed by conventional method.

[0087] The technical solutions of the present application are further illustrated by the following examples.

[0088] Example 1

[0089] The microbial composite film for improving the flavor and quality of ripened meat is prepared by scheme A (mixing microorganisms and water-soluble polymers and then spraying / spinning), which is used for dry ripening beef, and the specific steps are as follows:

[0090] 1.1 Preparation of bacteria blocking and air permeable layer

[0091] Poly lactic acid (PLA, Mw≈100 kDa) and polycaprolactone (PCL, Mw≈350 kDa) were blended at 4:6 (w / w), and then dissolved in a mixed solvent of dichloromethane / acetone (7:3, v / v) at a total concentration of 12 wt%, and electrospun at 25℃ and 45% humidity, with a voltage of 40 kV, a receiving distance of 20 cm, and a collection speed of 1.2 m / min. The electrospun product was vacuum dried at 60℃ for 12 h, and then sterilized by ultraviolet ozone for 30 min, to obtain a fibrous membrane with a grammage of 1.0±0.2 g / m 2 , a pore size of 0.80±0.13 μm, and a gas barrier property, which was recorded as a PLA bacterial barrier and gas permeable layer, and a scanning electron microscope image thereof is shown in FIG. 1. It can be seen that the fiber diameter is uniformly distributed and in a non-woven form, and the average pore size of the membrane is 0.80±0.13 μm, as analyzed by ImageJ analysis software. The membrane thickness was measured by a high-precision screw micrometer to be 18 μm, and the gas permeability was measured by GB / T 5453-1997 to be 35.2 mm / s (test pressure difference was 100 Pa). The interception efficiency for Bacillus pumilus spores was not less than 99.99% when the test flow rate was 2.8 L / min for 15 min. Figure 1

[0092] 1.2 Preparation of a functional bacteria loading layer

[0093] ① Preparation of a microbial bacteria solution: resuspend Thamnidium elegans spore suspension in PBS (pH=7.0) to 2×10 7 CFU / mL to obtain a microbial bacteria solution;

[0094] ② Preparation of a polymer solution: prepare a 10 wt% polyvinyl alcohol (PVA, Mw=89 kDa) aqueous solution, sterilize at 121℃ for 20 min, and obtain a PVA aqueous solution;

[0095] ③ Mixing: mix the microbial bacteria solution and the PVA aqueous solution at a volume ratio of 0.05:1, and magnetically stir for 10 min to obtain a mixed solution;

[0096] ④ Spraying: take a 10 cm×15 cm bacterial barrier and gas permeable layer, fix it on an electrostatic spraying platform, and uniformly spray the mixed solution on the surface of the bacterial barrier and gas permeable layer (the sprayed surface is the functional layer) at a rate of 0.2 mL / cm 2 , with a gun head distance of 8 cm from the membrane surface and a voltage of 12 kV. The loading density of the microorganisms in the functional bacteria loading layer is 5×10 2 CFU / cm 2 .

[0097] ⑤ Freeze-drying: freeze-dry the product obtained in step ④ at -55℃ and 10 Pa for 18 h to obtain a microbial composite membrane for improving the flavor and quality of cooked meat. ​

[0098] 1.3 Beef maturation verification

[0099] Take a 2.5 kg sirloin steak with a thickness of 8 cm (shear force of 68.3 N (Warner-Bratzler test method, the same below)), wipe the surface with 75% (volume fraction) alcohol, wrap it with the above-mentioned microbial composite film (functional layer facing the meat surface), and place it in a aging cabinet at 2℃, 80% RH (humidity), and 0.5 m / s wind speed. The control group was aged in an untreated environment.

[0100] The properties of the sirloin steak after 21 days are as follows:

[0101] After 21 days, the shear force of the sirloin steak in the microbial composite membrane group was 47.1 N, while that in the control group was 53.3 N. The shear force in the microbial composite membrane group decreased by 31%, while that in the control group decreased by 22%. After 21 days, the weight loss rate of the sirloin steak in the microbial composite membrane group was 13.8%, while that in the control group was 17.4%.

[0102] Flavor compounds: The samples were tested by gas chromatography-mass spectrometry (GC-MS), and the results are shown in Table 1.

[0103] Table 1. Content of several flavor compounds (μg / kg) after 14 days of aging.

[0104] odor molecules control group microbial complex membrane group hexane 73.5 101.2 2-butanone 69.1 74.7 3-hydroxy-2-butanone 186.4 204.8 2,5-dimethylpyrazine 80.6 98.3 3-methylbutanal 112.8 125.5

[0105] As shown in Table 1, the content of key odor molecules such as hexane, 2-butanone, 3-hydroxy-2-butanone, 2,5-dimethylpyrazine, and 3-methylbutanal in the microbial composite membrane group was higher than that in the control group, and the overall sensory score was significantly better than that of the control group.

[0106] Example 2

[0107] A microbial composite membrane for enhancing the flavor and quality of aged meat was prepared using Scheme B (first loading microorganisms, then immobilizing them with water-soluble polymers). This membrane was then used for the rapid aging of Parma ham. The specific steps are as follows:

[0108] 2.1 Preparation of the antibacterial and breathable layer

[0109] Thermoplastic polyurethane (TPU, Lubrizol 5702) was dissolved in DMF at a concentration of 15 wt%. Electrospinning was used to form a film with a needle-free electrospinning process at a voltage of 50 kV, a receiving distance of 20 cm, a collection speed of 3 m / min, a film thickness of 4.3 μm, and a pore size of 0.31 μm. The electrospun product was vacuum dried at 60℃ for 12 h and sterilized with ultraviolet ozone for 30 min. This film served as the antibacterial and breathable layer, denoted as the TPU antibacterial and breathable layer. Scanning electron microscopy images are shown below. Figure 2As shown, it can be seen that the fiber diameter is uniformly distributed and in non-woven form. The average pore size of the film was 0.53 ± 0.11 μm as determined using ImageJ analysis software. The film thickness was 23 μm as measured using a high-precision screw micrometer, and the air permeability was 26.3 mm / s (test pressure difference was 100 Pa) as determined using the method of GB / T 5453-1997; the interception efficiency for Bacillus pumilus spores was not less than 99.99% when the test flow rate was 2.8 L / min for 15 minutes.

[0110] 2.2 Preparation of functional bacteria loading layer

[0111] ①Yeast Debaryomyces hansenii and Staphylococcus xylosus were mixed at a ratio of 1:1, resuspended in PBS to 1 × 10 4 CFU / mL to obtain a microbial solution;

[0112] ②Bacterial solution spraying: the bacteria-resistant and air-permeable layer was laid flat, and air spraying was used with a spraying amount of 0.15 mL / cm 2 , and was left to dry at room temperature for 3 h;

[0113] ③Polymer immobilization: 8 wt% chitooligosaccharide (Mw = 50 kDa) solution was atomized and sprayed, with a spraying amount of 0.25 mL / cm 2 , and a film was formed with the assistance of hot air at 40°C. The loading density of the microorganisms in the functional bacteria loading layer was 1.8 × 10 2 CFU / cm 2 ;

[0114] ④Freeze-drying: the film obtained in step ③ was freeze-dried at -60°C and 15 Pa for 24 h to obtain a microbial composite film for improving the flavor and quality of cured meat.

[0115] 2.3 Ham curing verification

[0116] 3 kg of pig hind legs were taken, salted for 72 h in the traditional way, then desalted in running water for 24 h, the surface was wiped with 75% (volume fraction) alcohol, and the composite film was wrapped (the functional layer faced inwards), and was placed in a 15°C, 75% RH environment for curing. The control group was cured bare in the same environment.

[0117] After 60 days, the performance of the hams was as follows: the weight loss rate of the ham in the microbial composite film group was 12.1%, and the weight loss rate of the control group ham was 14.8%.

[0118] Sensory evaluation: the marble pattern of the ham cross-section in the microbial composite film group was more uniform, and free amino acids were tested using HPLC, and the results are shown in Table 2.

[0119] Table 2 Content of free amino acids (g / kg) after 60 days of curing

[0120]

[0121]

[0122] The results of Table 2 show that the free amino acid content of the microbial composite film group is higher than that of the control group.

[0123] Example 3

[0124] A microbial composite film for improving the flavor and quality of cooked meat was prepared using scheme C (first preparing active freeze-dried microbial powder, and then fixing with water-soluble polymer), and was used for dry aging of lamb legs. The specific steps are as follows:

[0125] 3.1 Preparation of the bacteria-resistant and air-permeable layer

[0126] Polyacrylonitrile (PAN, Mw = 70 kDa) and polyvinylidene fluoride (PVDF, Mw = 530 kDa) were dissolved in DMF at a concentration of 12 wt% (PAN:PVDF = 3:1, w / w), and needle electrospinning was performed at a voltage of 20 kV and a receiving distance of 12 cm, with a collection speed of 0.8 m / min. A fiber membrane with a thickness of 1.2 μm and a pore size of 0.18 μm was obtained, which was vacuum dried at 60°C for 12 h and ultraviolet ozone sterilized for 30 min to obtain the bacteria-resistant and air-permeable layer, which is denoted as Zein bacteria-resistant and air-permeable layer. The scanning electron microscope image is shown in FIG. 1, which shows that the fiber diameter is uniformly distributed and forms a non-woven form. The average pore size of the membrane was 0.48 ± 0.09 μm, as determined using ImageJ analysis software. The membrane thickness was 21 μm, as determined using a high-precision screw micrometer. The air permeability was 30.4 mm / s (test pressure difference was 100 Pa), as determined using the method of GB / T 5453-1997. The interception efficiency for Bacillus atrophaeus spores was not less than 99.99% when the test flow rate was 2.8 L / min for 15 minutes. Figure 3

[0127] 3.2 Preparation of the functional bacteria loading layer

[0128] ①Preparation of active freeze-dried microbial powder: the complex strain (lactic acid bacteria Lactobacillus sakei and yeast Debaryomyces hansenii at a ratio of 3:1) was cultured to the late logarithmic phase, centrifuged and collected, freeze-dried at -60°C and 15 Pa for 48 h, ground through a 100 mesh sieve, and active freeze-dried powder was obtained (microbial survival rate > 85%);

[0129] ②Powder spraying: the active freeze-dried microbial powder was uniformly deposited on the inside of the bacteria-resistant and air-permeable layer by electrostatic spraying (voltage was 25 kV, and powder feeding rate was 0.5 g / min), and the loading density of the microorganisms in the functional bacteria loading layer was controlled to be 8 × 10 2 ​CFU / cm 2 ;

[0130] 3. Polymer coating: The active freeze-dried microbial powder was coated with a 6 wt% gelatin / sodium alginate (mass ratio 7:3) solution by electrostatic spraying, with a spraying amount of 0.3 mL / cm 2 , to form a continuous coating layer;

[0131] 4. Freeze-drying: The active freeze-dried microbial powder was again freeze-dried at -55°C and 10 Pa for 20 h to ensure that the active freeze-dried microbial powder was fixed and there was no loss of activity, obtaining a microbial composite film for improving the flavor and quality of cooked meat.

[0132] 3.3 Leg verification

[0133] Take 2 kg of boneless leg (shear force is 33.8 N), wipe the surface with 75% (volume fraction) alcohol, tightly wrap with microbial composite film (functional layer facing in), and place in a 1°C, 85% RH aging box. The control group is aged bare under the same conditions.

[0134] The performance of the leg after 21 days is as follows:

[0135] Physicochemical indicators: The pH of the leg in the microbial composite film group decreased from 5.8 to 5.3, and the pH of the control group decreased to 5.4; the TVB-N (total volatile basic nitrogen) of the microbial composite film group was 11.2 mg / 100 g, which was significantly lower than the 14.7 mg / 100 g of the control group; the shear force of the microbial composite film was 24.0 N, with a 29% decrease in shear force; the shear force of the control group was 26.5 N, with a 21.6% decrease in shear force. The tenderness of the microbial composite film group was significantly improved.

[0136] Flavor: The rancid smell of the microbial composite film group was significantly reduced, and the samples were tested using gas chromatography-mass spectrometry (GC-MS), and the results are shown in Table 3.

[0137] Table 3: Content of several flavor substances after 14 days of aging (μg / kg)

[0138] control group microbial complex membrane group 2-methylbutanal 59.5 67.3 hexanal 922.4 1097.1 octanal 73.5 89.6 1-octen-3-ol 221.2 186.4 2-heptanone 13.5 24.3

[0139] The results of Table 3 show that the fat content of the leg in the microbial composite film group is significantly reduced, demonstrating the significant effect of aging on fat decomposition.

[0140] Comparative Example 1

[0141] 1. Preparation of a bacteria-resistant breathable film

[0142] The same as Example 3 "3.1", electrospun PAN:PVDF=3:1 (w / w) to obtain a fiber membrane with a thickness of 1.2 μm, a pore size of 0.18 μm, and a gas permeability of 30.4 mm / s (100 Pa), vacuum dried at 60°C for 12 h, and then sterilized by UV ozone for 30 min, ready for use.

[0143] 2 Leg wrapping and ripening

[0144] The same as Example 3 "3.3", 2 kg of boneless leg (shear force of 33.8 N, using the Warner-Bratzler test method) was wiped with 75% (volume fraction) alcohol, tightly wrapped with the antibacterial breathable film, and placed in a 1°C, 85% RH ripening box. The control group was ripened naked under the same conditions.

[0145] 3 Physicochemical indicators:

[0146] The pH of the leg ripened with the antibacterial breathable film in Comparative Example 1 decreased from 5.8 to 5.4; the TVB-N was 10.9 mg / 100 g; the shear force was 27.2 N, and the shear force decreased by 19.5%;

[0147] Flavor: The samples were tested by gas chromatography-mass spectrometry (GC-MS), and the results are shown in Table 4.

[0148] The single-layer antibacterial breathable film used in Comparative Example 1 was used to ripen the leg under the same conditions. Compared with the naked ripening control group, there was no significant change in pH, the shear force decreased slightly, and the flavor substance content was slightly less; compared with the leg ripened with the microbial composite film, there was no significant change in pH, the shear force decreased slightly, and the flavor substance content was significantly reduced. This is because the antibacterial film blocks all bacteria from participating in the ripening process and shields the effect of microorganisms on ripening. At the same time, the leg ripened with the single-layer antibacterial breathable film has less TVB-N content, because the antibacterial film shields the growth of harmful microorganisms and inhibits the production of TVB-N.

[0149] Comparative Example 2

[0150] 1. Preparation of functional bacteria-loaded film

[0151] The same as Example 3 "3.2", the substrate was a common medical polypropylene non-woven fabric (25 g / m2) without bacterial interception

[0152] ①Preparation of active freeze-dried microbial powder: the composite strain (lactic acid bacteria Lactobacillus sakei and yeast Debaryomyces hansenii at a ratio of 3:1) was cultured to the late logarithmic phase, centrifuged, freeze-dried at -60°C and 15 Pa for 48 h, ground through a 100 mesh sieve, and the active freeze-dried powder (microbial survival rate > 85%) was obtained;

[0153] ② Powder spraying: the active freeze-dried microbial powder was uniformly deposited on the non-woven fabric by electrostatic spraying (voltage 25 kV, powder feeding rate 0.5 g / min), and the loading density of the microorganism in the functional bacteria loading layer was controlled at 8 x 10 2 CFU / cm 2 ;

[0154] ③ Polymer coating: the active freeze-dried microbial powder was coated by electrostatic spraying using a 6wt% gelatin / sodium alginate (mass ratio 7:3) solution, and the spraying amount was 0.3 mL / cm 2 , forming a continuous coating layer;

[0155] ④ Freeze-drying: freeze-drying again at -55℃, 10 Pa for 20 h to ensure that the active freeze-dried microbial powder is fixed and there is no loss of activity, and obtain the functional bacteria loading film.

[0156] 2. Leg wrapping and ripening

[0157] As in Example 3, "3.3" paragraph, take 2 kg of boneless leg (shear force 33.8 N, using Warner-Bratzler test method), wipe the surface with 75% (volume fraction) alcohol, tightly wrap with the functional bacteria loading film, and place in a 1℃, 85% RH ripening box. The control group is ripened naked under the same conditions.

[0158] 3. Physicochemical indicators:

[0159] The pH of the leg of Example 2, which was ripened with the bacteria-resistant breathable film, decreased from 5.8 to 5.3; the TVB-N was 13.9 mg / 100 g; the shear force was 24.3 N, and the shear force decreased by 28.1%;

[0160] Flavor: the samples were tested by gas chromatography-mass spectrometry (GC-MS), and the results are shown in Table 4.

[0161] Table 4 Content of several flavor substances after 14 days of ripening (μg / kg)

[0162] comparative example 1 comparative example 2 2-methylbutanal 56.3 68.6 hexanal 883.2 1090.5 octanal 70.5 90.1 1-octen-3-ol 218.1 188.9 2-heptanone comparative example 1 comparative example 2 2-methylbutanal hexanal octanal 1-octen-3-ol 2-heptanone comparative example 1 comparative example 2 2-methylbutanal hexanal octanal 1-octen-3-ol 2-heptanone 11.8 24.5

[0163] The leg of mutton of the comparative example 2 is ripened under the same condition by using the functional bacteria loaded film. There is no obvious difference with the naked ripening control group, and the pH has no obvious change, the shear force decreases and the flavor substance content increases significantly; compared with the leg of mutton ripened by the microorganism composite film, the pH has no obvious change, and the shear force decreases and the flavor substance content are equivalent. This is the promotion effect of the beneficial microorganism in the functional bacteria loaded film on the ripening. Meanwhile, the TVB-N of the comparative example 2 is more than that of the leg of mutton ripened by the single layer bacteria blocking and air permeable film of the comparative example 1, and has no obvious difference with the naked ripening control group. This is due to the lack of the shielding effect of the bacteria blocking film, and the harmful microorganism breeds on the meat surface. However, the TVB-N content of the comparative example 2 is slightly lower than that of the naked ripening control group. This is due to the fact that the beneficial microorganism loaded in the functional bacteria loaded film forms a dominant bacterial population on the meat surface, and the harmful microorganism breeds relatively less.

[0164] The above comparative examples prove the interception effect of the bacteria blocking and air permeable film in the microorganism composite film on the harmful bacteria, and the promotion effect of the microorganism loaded layer on the meat ripening. The two improve the quality of the meat ripening.

[0165] The above, only for the preferred specific embodiments of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art in the technical range disclosed by the present application, can easily think of the changes or replacements, should be covered in the protection scope of the present application.

Claims

1. A microbial composite membrane for enhancing the flavor and quality of cooked meat, characterized in that, The microbial composite membrane includes a bacterial barrier and air-permeable layer and a functional bacterial loading layer; The antibacterial and breathable layer is used to block external microorganisms from entering and prevent harmful bacteria or fungi from multiplying on the surface of meat. The functional microbial support layer is loaded with freeze-dried microorganisms that are beneficial to the cooking of meat.

2. The microbial composite membrane for enhancing the flavor and quality of cooked meat according to claim 1, characterized in that, The load density of the freeze-dried microorganisms in the functional bacterial support layer is 10–1000 CFU / cm³. 2 .

3. The microbial composite membrane for enhancing the flavor and quality of cooked meat according to claim 2, characterized in that, The freeze-dried microorganisms are selected from freeze-dried fungi, freeze-dried fungal spores, or freeze-dried bacteria.

4. The microbial composite membrane for enhancing the flavor and quality of cooked meat according to claim 1, characterized in that, The pore size of the antibacterial and breathable layer is 0.1–0.8 μm, the thickness is 0.3–10 μm, and the air permeability is not less than 5 mm / s when the test pressure difference is 100 Pa.

5. The microbial composite membrane for enhancing the flavor and quality of cooked meat according to claim 4, characterized in that, The raw material for preparing the antibacterial and breathable layer is a non-water-soluble polymer.

6. The microbial composite membrane for enhancing the flavor and quality of cooked meat according to claim 5, characterized in that, The non-water-soluble polymer is selected from at least one of polylactic acid, polycaprolactone, polyurethane, polystyrene, polyethersulfone, polyvinylidene fluoride, polyimide, zein, and polyacrylonitrile.

7. The microbial composite membrane for enhancing the flavor and quality of cooked meat according to claim 1, characterized in that, The functional bacteria in the functional bacterial load layer were immobilized using a water-soluble polymer.

8. The microbial composite membrane for enhancing the flavor and quality of cooked meat according to claim 7, characterized in that, The water-soluble polymer is selected from at least one of polyvinyl alcohol, polyethylene oxide, vinylpyrrolidone, sodium alginate, carboxymethyl cellulose, corn gluten, starch, guar gum, and gum arabic.

9. A method for preparing a microbial composite membrane for improving the flavor and quality of cooked meat according to any one of claims 1 to 8, characterized in that, Includes the following steps: After mixing microbial bacterial solution with water-soluble polymer solution, the mixture is loaded onto the inner side of the antibacterial and breathable layer by spraying or electrospinning, and then freeze-drying is performed to obtain the microbial composite membrane used to improve the flavor and quality of cooked meat. Alternatively, the microbial liquid can be sprayed onto the inner side of the antibacterial and breathable layer, and then the microorganisms can be fixed by spraying or electrospinning with a water-soluble polymer solution. Finally, the microbial composite membrane used to improve the flavor and quality of cooked meat can be obtained. Alternatively, the microbial liquid can be pre-freeze-dried into powder, and the powder can be distributed on the inner side of the antibacterial and breathable layer by electrostatic spraying. Then, the microorganisms can be fixed by spraying with a water-soluble polymer solution or by electrospinning to obtain the microbial composite membrane used to improve the flavor and quality of cooked meat.

10. The application of a microbial composite membrane for enhancing the flavor and quality of cooked meat as described in any one of claims 1 to 8 in the cooking of meat.

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