Mozzarella cheese and preparation method thereof

Abstract

The invention relates to the field of food, and particularly discloses Mozzarella cheese and a preparation method thereof.The method comprises the steps that whole milk is subjected to degreasing treatment and ultrafiltration treatment, and raw milk is obtained; performing sterilization treatment and pre-acidification treatment on the raw milk to obtain acidified feed liquid; inoculating the acidified feed liquid with a first leavening agent, and carrying out pre-fermentation treatment to obtain a pre-fermentation product; sequentially performing curding treatment, whey removal treatment, crushing treatment, blanching stretching treatment, cooling treatment and salting treatment on the pre-fermented product to obtain a salted product; and inoculating the surface of the salted product with a second leavening agent to obtain the mozzarella cheese. By means of the method, the production efficiency of the mozzarella cheese can be improved, the prepared cheese can form a casein-calcium matrix structure with ideal melting and drawing performance and storage stability, and meanwhile the flavor richness of the cheese is effectively enhanced.

Description

Technical Field

[0001] This application relates to the food industry, and more specifically, to mozzarella cheese and its preparation method. Background Technology

[0002] Low-moisture partially defatted (LMPS) mozzarella cheese is a core ingredient in pizza and related baked goods, and its consumption continues to grow. The excellent melting, stringing, and browning properties exhibited by this type of cheese upon heating are primarily attributed to its unique casein matrix structure and proper calcium balance. These functional properties are key to determining the quality of the final food product. To improve production efficiency and reduce energy consumption and costs, the dairy industry is increasingly adopting membrane filtration technology to produce concentrated milk as a cheese raw material. Increasing the casein content in raw milk can significantly improve batch yield, and studies have shown that LMPS cheese produced in this way typically has a denser protein network, higher firmness, and better functional stability during storage. However, this efficient industrialization path also brings new technical challenges: First, concentrated milk alters fermentation kinetics and the coagulation environment, potentially leading to insufficient flavor development and a lack of complexity and richness in the final product compared to traditional cheeses; second, specific process parameters used to control the texture and melting properties of concentrated milk cheese (such as lower whey pH) may further inhibit the activity or metabolic pathways of microorganisms involved in flavor formation. Currently, most improvement solutions in the industry focus on a single dimension: either improving texture by optimizing concentration and acidification processes, or attempting to enhance flavor by adjusting starter cultures. However, these two approaches often conflict. For example, advancing or increasing the amount of starter culture to enhance flavor may interfere with the curdling process of concentrated milk, damaging functional texture; while over-optimizing process parameters for texture may limit flavor generation.

[0003] Therefore, existing technologies lack a comprehensive solution that can systematically and collaboratively address the core issue of "how to ensure and enhance product flavor while improving production efficiency and functional texture of high casein concentrate". Summary of the Invention

[0004] This application aims to at least partially address the technical problems existing in the prior art. To this end, this application proposes a mozzarella cheese and a method for preparing the same, which can improve the production efficiency of mozzarella cheese, enable the obtained cheese to form a casein-calcium matrix structure with ideal melting, stringing properties and storage stability, and effectively enhance the flavor richness of the cheese.

[0005] This application is based on the inventor's following discoveries: The inventors discovered through experiments that while using ultrafiltration to prepare high casein concentrate (or raw milk) can effectively improve cheese production efficiency, it alters the fermentation microenvironment, thereby inhibiting the formation of flavor compounds and resulting in a monotonous flavor in the finished cheese. Furthermore, the industry currently predominantly employs a single-inoculation method, where all starter cultures (whether single or compound strains) are added all at once before coagulation. Their core function is merely to complete basic fermentation processes such as acid production and coagulation, making precise flavor control difficult. Specifically, in the traditional single-inoculation method, the formation of flavor compounds relies entirely on the metabolic processes of the microorganisms in the complex and unstable fermentation environment of the early stages. This process is poorly controllable, and once flavor is formed, it is difficult to specifically enhance it in later stages, ultimately resulting in a finished cheese lacking the flavor complexity and richness of products made using traditional methods.

[0006] To address the aforementioned technical deficiencies, the inventors innovatively proposed a phased, time-series control process: "acid-producing curd with a primary starter culture + targeted aroma development through secondary inoculation." After the core processes of curdling, whey removal, pulverization, and stretching are completed, a specific salt-tolerant flavor starter culture (such as *Lactococcus lactis* subsp. *milk fat*) is added a second time to the pre-formed cheese matrix before salting (dry salt stage) or vacuum packaging. This innovative process allows the flavor starter culture to effectively avoid the unfavorable environment of high acidity and high concentration of the initial milk matrix, focusing instead on the synthesis of flavor compounds during the post-ripening stage within the already stable cheese matrix. This results in the targeted production of key flavor compounds such as diacetyl (also known as diacetyl) and short-chain fatty acids, thereby achieving proactive, precise, and enhanced control over the cheese flavor.

[0007] Therefore, in one aspect of this application, a method for preparing mozzarella cheese is proposed. According to an embodiment of this application, the method includes: defatting and ultrafiltration of whole milk to obtain raw milk; sterilizing and pre-acidifying the raw milk to obtain an acidified liquid; inoculating the acidified liquid with a first fermenting agent for pre-fermentation to obtain a pre-fermented product; subjecting the pre-fermented product to a series of processes including curdling, whey removal, pulverization, heat treatment and stretching, cooling, and salting to obtain a salted product; and inoculating the surface of the salted product with a second fermenting agent to obtain the mozzarella cheese. Thus, the preparation method of this application can fully utilize the production efficiency advantages of high casein concentrate, resulting in mozzarella cheese that not only possesses excellent functional properties such as melting properties, stringiness, and storage stability, but also boasts a flavor richness, complexity, and pleasantness comparable to or even superior to products made using traditional methods.

[0008] According to embodiments of this application, the above-described method for preparing mozzarella cheese may further include the following additional technical features: In some embodiments, the ultrafiltration treatment is performed using an ultrafiltration membrane system. In some embodiments, the inlet pressure of the ultrafiltration membrane system is 350-390 kPa, for example, 350 kPa, 360 kPa, 370 kPa, 380 kPa, 390 kPa, or any range of these values. In some embodiments, the outlet pressure of the ultrafiltration membrane system is controlled at 150-190 kPa, for example, 150 kPa, 160 kPa, 170 kPa, 180 kPa, 190 kPa, or any range of these values. By controlling the inlet and outlet pressures within the above ranges, stable transmembrane pressure can be maintained, thereby ensuring that the membrane permeation flux and separation accuracy remain constant. This ensures that concentrated milk produced in different batches exhibits high uniformity in key indicators such as protein content (e.g., approximately 5.8%), calcium content, and mineral composition, providing precisely reproducible standardized raw materials for subsequent cheese production, thereby improving the overall efficiency of cheese production and the stability of product quality.

[0009] According to embodiments of this application, the casein content in the raw milk is 2.5% to 4.0% by mass. For example, it can be 2.5%, 2.7%, 3.0%, 3.2%, 3.5%, 3.7%, 4.0%, or any range of the above values. Casein is a core component of the cheese matrix. Increasing the casein content in the raw milk directly increases the dry matter yield per batch of cheese, reduces raw material loss caused by whey discharge, and meets the cost reduction and efficiency improvement requirements of large-scale industrial production. Simultaneously, it lays the foundation for constructing a denser casein-calcium network structure that facilitates melting and stringing.

[0010] In some embodiments, the casein content in the raw milk is 3.5% to 4.0% by mass. Therefore, standardizing the raw milk to a casein content of 3.5%-4.0% through ultrafiltration can significantly improve production efficiency while more stably constructing a cheese protein matrix with ideal melting and stringing properties, reducing quality differences caused by raw material fluctuations.

[0011] According to an embodiment of this application, the mass ratio of casein to fat in the raw milk is 1:(1~1.1). For example, it can be 1:1, 1:1.02, 1:1.05, 1:1.07, 1:1.1, 1:1, etc., or any range of the above values. By controlling the casein to fat ratio within the above range, it is possible to ensure that the cheese has ideal melting properties, stringiness, and browning ability when heated, avoiding problems such as excessive oil release or a texture that is too hard or too soft after melting, thus meeting the needs of baking applications such as pizza.

[0012] According to embodiments of this application, the protein content in the raw milk is 5% to 6% by mass. For example, it can be 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, or any range of the above values. This ensures that the raw milk contains sufficient casein.

[0013] According to an embodiment of this application, the sterilization treatment is performed at a temperature of 72-74°C for 14-16 seconds. Exemplarily, the temperature can be 72°C, 73°C, 74°C, etc., and the time can be 14 seconds, 15 seconds, 16 seconds, etc. This facilitates the thorough elimination of unwanted microorganisms and reduces nutrient loss.

[0014] According to an embodiment of this application, before subjecting the sterilized raw milk to the pre-acidification treatment, the sterilized raw milk is cooled to 5-10°C, for example, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. This facilitates the chelation of the acidifying agent with calcium ions in the raw milk, forming calcium salt precipitates.

[0015] According to embodiments of this application, the pre-acidification treatment is carried out using an acidifier, which includes one or more of lactic acid, citric acid, acetic acid, malic acid, hydrochloric acid, phosphoric acid, or glucono-δ-lactone. According to embodiments of this application, the acidifier is lactic acid or citric acid because it can effectively lower the pH of the raw milk to a target value (e.g., pH 6.0) while promoting the dissolution of calcium from casein micelles, which is beneficial for the subsequent formation of an ideal cheese texture. For high casein concentrate, lactic acid pre-acidification can more significantly reduce the calcium content, thereby further improving the texture and softness of low-fat products.

[0016] In some embodiments, the pre-acidification treatment is carried out using an aqueous lactic acid solution, which is prepared by mixing 88% food-grade lactic acid and water at a mass ratio of 1:4. This achieves the following technical effects: (1) moderately reducing the pH of the raw milk to create a suitable acidic environment for subsequent fermentation and curdling; (2) promoting the dissolution of colloidal calcium phosphate, converting some insoluble calcium into soluble calcium, and reducing the content of insoluble calcium as a casein crosslinking material in the final cheese; (3) regulating the dissociation state of casein micelles, changing the proportion of soluble micelle casein, and optimizing the gel network structure induced by rennet; (4) improving the textural properties of the final cheese, reducing hardness, increasing melting and stretchability, and maintaining good functional performance during storage.

[0017] According to an embodiment of this application, the pre-acidification treatment time is 40-50 minutes. For example, it can be 40 minutes, 42 minutes, 45 minutes, 47 minutes, 50 minutes, or any range of the above values. This allows the pH of the acidified solution to be 5.5-6.5, for example, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, etc. According to an embodiment of this application, the first fermenting agent includes one or more of *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, *Lactobacillus helveticus*, *Lactobacillus casei*, or *Streptococcus lactis*. Therefore, by using a first fermenting agent selected from the above types, it is possible to moderately produce acid during the pre-fermentation stage, adjusting the pH of the raw milk to a suitable range for coagulation. This ensures that the acidity of the raw milk is consistent when rennet is subsequently added, avoiding problems such as excessively fast or slow coagulation speed and uneven coagulation texture caused by acidity fluctuations.

[0018] According to an embodiment of this application, the casein content in the raw milk is 2.5% by mass. Based on 100 kg of the acidified liquid, the amount of the first starter culture added is 15-25 g. For example, the amount of the first starter culture added can be 15 g, 17 g, 20 g, 22 g, 25 g, etc., or can be any range of the above values. Casein is one of the core substrates for fermentation. The higher the casein content of the raw milk, the more starter culture is required. Increasing the inoculum amount according to the casein ratio allows the viable cell count of the starter culture to be matched with the substrate concentration, avoiding the problems of slow acid production due to insufficient starter culture or excessive acid production due to excessive starter culture.

[0019] According to embodiments of this application, the casein content in the raw milk is less than 3.5% by mass, and the pre-fermentation time is 50-70 minutes; the casein content in the raw milk is not less than 3.5% by mass, and the pre-fermentation time is 70-80 minutes. For example, if the casein content in the raw milk is less than 3.5% by mass, the pre-fermentation time can be 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, etc., or any range of the above values. If the casein content in the raw milk is not less than 3.5% by mass, the pre-fermentation time can be 70 minutes, 72 minutes, 75 minutes, 77 minutes, 80 minutes, etc., or any range of the above values. Therefore, raw milk with high casein content has a denser matrix, which slows down the metabolism of the starter culture, resulting in a slower natural acid production rate. By extending the pre-fermentation time, the difference in acid production rate can be compensated for, allowing raw milk with different casein contents to reach similar pH values ​​at the end of pre-fermentation.

[0020] In some embodiments, when the casein content in the raw milk is 3.5%, the pre-fermentation time is preferably 70 minutes; when the casein content in the raw milk is 4.0%, the pre-fermentation time is preferably 75 minutes. This optimization ensures that nearly consistent acid production curves can be obtained for raw milk of different concentrations, creating more stable conditions for subsequent steps. It should be noted that the addition of rennet is strictly calculated based on this standardized total casein content.

[0021] According to an embodiment of this application, the temperature of the pre-fermentation treatment is 33~37℃. For example, it can be 33℃, 34℃, 35℃, 36℃, 37℃, etc., or it can be any range of the above values.

[0022] According to embodiments of this application, the second fermenting agent includes *Lactococcus lactis* subsp. *lactofat*. Through extensive experimentation, the inventors discovered that *Lactococcus lactis* subsp. *lactofat* is a preferred strain suitable for the secondary inoculation requirements of this application. It not only possesses excellent salt tolerance, adapting to the high-salt environment of the cheese matrix after salting, and successfully initiating post-fermentation in the formed cheese matrix, but also, because it does not participate in the curdling process, does not interfere with the previously constructed casein-calcium network structure. Furthermore, its core metabolic advantage lies in its ability to synthesize large quantities of key flavor compounds such as diacetyl and short-chain fatty acids, imparting a rich creamy and nutty aroma to the cheese, thereby precisely compensating for the flavor monotony caused by the high casein concentrate process.

[0023] According to an embodiment of this application, the concentration of the second fermenting agent on the surface of the salted product is 10. 6 ~10 7 CFU / g. For example, it can be 10. 6 CFU / g, 2×10 6 CFU / g, 5×10 6 CFU / g, 7×10 6 CFU / g, 10 7 CFU / g, etc., or a range consisting of any of the above values. Wherein, 10 6 ~10 7 The CFU / g concentration refers to the initial viable cell concentration achieved in the salted product (top 0-5mm region) after inoculation with the second starter culture and before packaging. Therefore, by controlling the concentration of the second starter culture within this range, the microorganisms in the second starter culture can rapidly colonize the cheese matrix and form a dominant microbial community. This avoids both excessively low concentrations leading to weak microbial competitiveness and insufficient flavor compound synthesis, and excessively high concentrations causing over-fermentation, resulting in off-flavors or damaging the cheese texture. This concentration is a key process parameter to ensure that the flavor bacteria effectively initiate post-fermentation and produce sufficient flavor compounds during the subsequent maturation process.

[0024] According to an embodiment of this application, the concentration of the second fermenting agent on the surface of the salted product is 5 × 10⁻⁶. 6 CFU / g. Therefore, at this concentration, the post-fermentation process can be initiated and dominated more efficiently, maximizing the efficiency of flavor compound synthesis.

[0025] According to an embodiment of this application, the curdling process includes: adding rennet to the pre-fermented product, stirring, allowing it to stand, and then cutting it into pieces. According to an embodiment of this application, based on the total casein content, the mass-to-volume ratio of casein to rennet is 1 kg:(5~6) mL; the enzyme activity of the rennet is ≥190 IMCU / ml. The substrate for rennet is casein, and adding it according to the above ratio ensures that the enzyme dosage precisely corresponds to the casein content in the raw milk. This avoids problems such as slow curdling and loose curds due to insufficient enzymes caused by high casein content, or excessive curdling and hard curds due to excessive enzymes. It should be noted that since the total casein content in the prepared raw milk does not change during subsequent experiments, the total casein content here can be calculated based on the total casein content in the raw milk.

[0026] According to embodiments of this application, the settling time is 25-35 minutes. For example, it can be 25 minutes, 27 minutes, 30 minutes, 32 minutes, 35 minutes, or any range of the above values. The settling process allows rennet to fully contact casein. Therefore, by controlling the settling time within the above range, it is possible to promote the cross-linking of casein micelles to form a continuous gel network, thereby improving the melting properties, stringiness, and storage stability of the cheese.

[0027] According to embodiments of this application, after cutting into blocks, the cheese is left to stand for 10-20 minutes. For example, this could be 10 minutes, 12 minutes, 15 minutes, 17 minutes, 20 minutes, or any range of these values. Cutting breaks the gel network of the curd. By allowing it to stand for the aforementioned time, some free whey can be released, making the curd firmer and more resilient from a loose, cut state. This prevents the curd from breaking apart during subsequent whey removal, reduces casein loss, and thus improves the cheese's melting properties, stringiness, and storage stability.

[0028] According to an embodiment of this application, the whey removal process includes: heating the curd product, and removing the whey when the pH of the heated product is 5.6-5.7. The pH value directly affects the degree of cross-linking of casein micelles. When the pH drops to 5.7, the casein-calcium network has formed a firm and resilient stable structure, making the curd particles less prone to breakage due to external forces (such as stirring or filtering during whey removal), thus reducing casein loss with the whey and ensuring the yield of dry matter in cheese. If the pH is too high, the casein cross-linking is insufficient, and the curd particles are loose and brittle, significantly increasing raw material loss.

[0029] According to an embodiment of this application, the temperature of the heat treatment is 35~40℃. For example, it can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc., or any range of the above values. According to an embodiment of this application, the heat treatment time is 15~25 minutes. For example, it can be 15 minutes, 17 minutes, 20 minutes, 22 minutes, 25 minutes, etc., or any range of the above values. Therefore, through heat treatment, the free whey in the curd product can be more easily separated from the curd particles, which can improve the efficiency of subsequent whey removal and control the cheese moisture content within the suitable range for low-moisture mozzarella cheese, ensuring the storage stability and heating melting performance of the finished product.

[0030] According to an embodiment of this application, the whey removal time is 25-35 minutes. For example, it can be 25 minutes, 27 minutes, 30 minutes, 32 minutes, 35 minutes, or any range of the above values. Therefore, by controlling the whey removal time within the above range, it is possible to avoid excessive moisture causing the cheese to release oil and lose its stringiness during melting, and also to prevent excessive moisture causing the cheese to become dry, easily broken, and prone to stretching, while ensuring the storage stability of the finished product.

[0031] According to the embodiments of this application, the pH of the curd after whey removal treatment is 5.50~5.65. The inventors discovered through experiments that controlling the pH of the curd within the above range allows the curd to remain soft and non-sticky, yet tough and unbreakable under stretching conditions, and can be smoothly stretched into a dense and uniform cheese matrix. If the pH is too high, the curd has excessive water retention and is too sticky, easily clumping and sticking together during stretching, and cannot form a continuous fibrous structure. If the pH is too low, the curd is over-acidified, dry, hard, and brittle, and is extremely easy to break during stretching, directly losing the core stringy properties of mozzarella cheese.

[0032] It should be noted that the statement "the pH of the curd after whey removal is 5.50~5.65" refers to the initial pH value of the curd when the whey removal process is completed. Because sufficient primary starter remains inside the curd, and the temperature conditions at this time are still suitable for its metabolic activity, the starter will not stop producing acid after the whey removal process ends. It will continue to decompose lactose and generate lactic acid, and the continuous accumulation of lactic acid will drive the pH value of the curd to gradually decrease.

[0033] According to an embodiment of this application, the pulverization process includes pulverizing the whey-drained curd at a pH of 5.10 to 5.40. Exemplarily, the pH can be 5.10, 5.15, 5.20, 5.25, 5.30, 5.35, 5.40, or any range of the above values. According to an embodiment of this application, the pulverization process includes pulverizing the whey-drained curd at a pH of 5.15 to 5.25. Through extensive experimental verification, the inventors discovered that under this specific low pH environment, the dissolution and redistribution of calcium ions can be more precisely controlled, thereby optimizing the cross-linking density of the casein network. This acidity control, combined with the preferred high casein content, creates a synergistic effect, resulting in cheese with longer string length, better melt flowability, and a more supple texture.

[0034] According to an embodiment of this application, the hot stretching treatment includes stretching the curd obtained from the crushing treatment in hot water at 70-80°C for 5-10 minutes. This enables the cheese to possess excellent properties such as longer cheese pull length, better fluidity after melting, and a more supple texture.

[0035] According to an embodiment of this application, the cooling process includes placing the product obtained from the hot stretching treatment in a cold water bath for 50-70 minutes until the internal temperature of the product is below 38°C. This allows the cheese to possess excellent properties such as longer cheese pull length, better fluidity after melting, and a more supple texture.

[0036] According to an embodiment of this application, the salting treatment includes placing the product obtained from the cooling treatment in an 18-22% by mass salt solution for 3-5 hours. This allows the salt to bind with the moisture in the cheese, reducing the cheese's water activity, further stabilizing the water-holding capacity of the casein-calcium network, and improving the cheese's stringiness, melting properties, and stability.

[0037] According to embodiments of this application, the maturation process is carried out at a temperature of 2-6°C for 15-20 days. This allows the second fermenting agent to continuously decompose nutrients in the substrate, synthesizing key flavor compounds such as diacetyl and short-chain fatty acids, thus imparting a rich creamy and nutty aroma to the cheese and transforming the originally basic salty cheese into a complex and layered flavor profile.

[0038] In summary, the method of this application has the following advantages: 1) Improved and stable product functionality and texture: By fully utilizing the production efficiency advantages brought by high casein concentrate, and by limiting the casein starting point and matching the curdling, milk removal, and crushing pH control processes, it is possible to more stably produce cheese that maintains excellent stringiness, high elasticity, and uniform melting during refrigerated storage, and significantly improve the batch consistency of its pizza baking performance (such as uniformity of charred spots and fat precipitation); 2) Enhanced flavor development intensity and speed: By performing secondary inoculation, the content of key flavor substances such as diacetyl and short-chain fatty acids in the cheese is increased by more than 20%, thereby producing a more vivid, full-bodied, and layered cream and nutty flavor, resulting in higher sensory evaluation scores; 3) Enhanced process efficiency and operability: The clearly defined optimal range of casein content and the corresponding pre-fermentation time simplify process decisions in production, reduce operational difficulty, and make this integrated process easier to standardize and scale up in industrial production, thereby improving the overall efficiency and reliability of the production line.

[0039] In another aspect of this application, a mozzarella cheese is proposed. According to an embodiment of this application, the mozzarella cheese is obtained by the aforementioned method for preparing mozzarella cheese. The mozzarella cheese of this application possesses excellent melting properties, stringiness, and storage stability due to its casein-calcium matrix structure; compared to products made using traditional methods, it also exhibits a richer, more intense, and more natural flavor profile.

[0040] The differences between this application and the prior art are as follows: 1. The fermentation process sequence and purpose are fundamentally different: "Secondary inoculation" is introduced for targeted flavor enhancement. Existing technologies employ a single-stage inoculation method before curdling, where the starter culture only completes the basic process of acid-producing curdling. Flavor formation is affected by the complex environment in the early stages, has poor controllability, and is difficult to enhance in the later stages. This application innovatively adopts a phased process of "main starter culture for acid-producing curdling + secondary inoculation for targeted aroma development," adding a salt-tolerant flavor starter culture after the cheese matrix is ​​formed. This allows the starter culture to focus on synthesizing post-ripening flavor substances in a stable matrix, achieving proactive, precise, and enhanced control over the cheese flavor.

[0041] 2. Deepening the application objectives of concentrated milk: from "improving efficiency" to "synergistic design of function and flavor" In existing technologies, the application of ultrafiltration concentrate milk focuses only on improving cheese production efficiency and increasing dry matter output. Process adjustments are also limited to adapting concentrate milk to produce cheese with acceptable texture, and the resulting flavor loss is often tacitly accepted. However, this application selects concentrate milk with a specific casein content (e.g., 2.5%-4.0%), using it not only as a tool to improve production efficiency but also as the foundation for constructing a high-quality functional matrix for cheese. By controlling key processes such as pre-acidification and pulverization pH, a protein-calcium network is actively constructed to facilitate excellent melting and stringing properties of cheese. At the same time, the "secondary inoculation" technology is used to compensate for the flavor defects caused by the concentrate milk process, ultimately achieving synergistic optimization of cheese production efficiency, product functional characteristics, and flavor performance.

[0042] 3. Refinement of acidification and texture control strategies: Synergistic control of key process points Existing technologies for acidification control in cheese production largely rely on the natural acid production of starter cultures or simple chemical pre-acidification. The pH parameters for key processes such as whey removal and grinding are often used within a wide or fixed range, without precise linkage and control with the casein content of the concentrated milk. This application, however, constructs a synergistic process control chain across all stages. It not only adjusts the pH of the raw milk to 5.5–6.5 through pre-acidification and sets the whey removal trigger point at approximately 5.6–5.7, and dynamically adjusts the fermentation time based on casein content, but also uses grinding pH as a core control variable, preferably controlled within the lower range of 5.10–5.40. Through the synergistic effect of this parameter and high casein content, it optimizes calcium ion dissolution, thereby improving the texture and melting properties of the cheese. This type of precise, multi-stage synergistic control method is rarely systematically demonstrated or applied in existing technologies.

[0043] 4. Different paths to achieving product quality: not relying on non-dairy additives. Existing technologies often require the addition of non-dairy components such as stabilizers, emulsifiers, colloids, and flavorings to improve the texture, stability, and flavor of low-fat or concentrated cheese. However, this application achieves both excellent textural functionality and a rich, naturally fermented flavor in cheese without the addition of any functional food additives. This makes the finished product more in line with the "clean label" consumer trend and gives it higher market value and a differentiated competitive advantage.

[0044] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Detailed Implementation

[0045] The embodiments of the technical solution of this application are described in detail below. These embodiments are only used to more clearly illustrate the technical solution of this application, and are therefore merely examples and should not be used to limit the scope of protection of this application.

[0046] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0047] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​1 and 2 are listed, and if maximum range values ​​3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers, and ranges defined in this way can include endpoints a and b. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0048] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0049] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0050] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0051] This application discloses a method for preparing mozzarella cheese, the method comprising: 1. Raw milk preparation Whole milk was used as raw material, and partially defatted to reduce the fat content to approximately 2.35%–3.85%. Subsequently, the partially defatted milk was concentrated using a spiral-wound ultrafiltration membrane system with a molecular weight cutoff of 10 kDa at approximately 4°C. The system throughput was controlled at approximately 6.4 L / min. By adjusting the inlet pressure (approximately 350–390 kPa), outlet pressure (approximately 150–180 kPa), and pressurization (approximately 180–200 kPa), a retentate with a concentration factor of 2.2 and a protein content of approximately 5%–6% (w / w) was finally obtained. This ultrafiltration retentate was precisely blended with ultrafiltration permeate and butter (at a casein to fat ratio of 1:(1–1.1)) to standardize the raw milk used for cheese production to a target casein content of 2.5%–4.0% (w / w).

[0052] 2. Sterilization and pre-acidification The standardized raw milk is pasteurized at 72-74°C for 14-16 seconds. Subsequently, the pasteurized milk is cooled to approximately 7°C and pre-acidified (PA) with a food-grade lactic acid aqueous solution (88% lactic acid diluted at a 1:4 ratio) at a constant rate for 40-50 minutes to adjust the pH of the raw milk to 5.5-6.5.

[0053] 3. Pre-fermentation The pre-acidified raw milk is heated to 33°C. The first starter culture is added proportionally according to the casein content of the raw milk. For raw milk with a casein content of 2.5%, the addition standard is (15~25) g / 100 kg (first starter culture / raw milk). For raw milk with a higher casein content, the inoculum amount is increased accordingly based on the casein ratio. After adding the starter culture, the raw milk is pre-fermented. To achieve similar acid production rates for raw milk with different casein contents, raw milk with a casein content below 3.5% is pre-fermented for 50~70 minutes (e.g., 60 minutes), while raw milk with a casein content of 3.5% and above is pre-fermented for 70~80 minutes (e.g., 75 minutes).

[0054] 4. Curd After pre-fermentation, calculate and add microbial rennet (activity ≥190 IMCU / ml, MICROLANT® Supreme 200 NB, ChrHansen) at a ratio of 5.5~6.0 ml per kilogram of casein, based on the actual total casein content in the raw milk. Gently stir until homogeneous and let the curd stand for 25~35 minutes. Once the curd reaches the appropriate firmness, cut it with a 2.54 cm wide knife and let it stand to heal for 10~20 minutes.

[0055] 5. Cooking and draining whey Subsequently, the curd particles are cooked at approximately 35–40°C for 15–25 minutes. When the pH value of the curd particles drops to approximately 5.60–5.70, whey is slowly drained, a process that lasts approximately 25–35 minutes. At the end of the whey drainage, the pH value of the curd particles is approximately 5.50–5.65.

[0056] 6. Crushing and stretching The curd was cut into blocks and divided into two parts, which were then pulverized when the pH value of the curd dropped to 5.10-5.40. The pulverized curd particles were then mechanically stretched in hot water at 70-80°C for 5-10 minutes, so that the outlet temperature of the curd reached about 60°C, until a smooth and uniform cheese mass was formed.

[0057] 7. Shaping, Cooling and Salting The stretched hot cheese dough is extruded into shape (e.g., a 9×9×27 cm rectangular mold) and then immersed in a cold water bath to cool for 50–70 minutes, allowing the internal temperature to drop below 38°C. Afterward, the cheese blocks are placed in a 4°C brine solution with a concentration of 18%–22% (w / w) for 3–5 hours to salt.

[0058] 8. Second inoculation and packaging After salting and draining, before packaging, evenly spray or coat the surface of the cheese blocks with a suspension of the selected second leavening agent. The amount of the flavor leavening agent added should be such that it reaches 10% of the cheese matrix. 6 ~10 7 The initial concentration was set at CFU / g. The cheese cubes were then immediately sealed in high-barrier packaging bags using a vacuum packaging machine.

[0059] 9. Refrigerated Maturation and Testing Vacuum-packed cheese blocks were refrigerated at 4°C for maturation. After 15-20 days of storage, samples were taken and evaluated according to standard methods (e.g., 9-inch pizza dough, approximately 20 cm; 50 g tomato sauce; 100 g shredded cheese; baked at 250°C for 5 minutes using a chain oven, followed by cooling for 90-120 seconds until the sample temperature reached 70-80°C). Key performance indicators such as melting properties, stringiness, charring, texture, and flavor were assessed.

[0060] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0061] Example 1 1. Raw Material Processing: Whole milk was used as the raw material and partially defatted to reduce the fat content to approximately 2.38%. Subsequently, a spiral-wound ultrafiltration membrane system with a molecular weight cutoff of 10 kDa was used to concentrate the partially defatted milk at approximately 4°C. The system throughput was controlled at approximately 6.4 L / min. By adjusting the inlet pressure (approximately 375 kPa), outlet pressure (approximately 170 kPa), and pressurization (approximately 190 kPa), a retentate with a concentration factor of 2.2 and a protein content of approximately 5.8% (w / w) was finally obtained. This ultrafiltration retentate was then precisely blended with ultrafiltration permeate and butter (at a casein to fat ratio of 1.05) to standardize the raw milk used for cheese production to a target casein content of 2.5% (w / w).

[0062] 2. Sterilization and pre-acidification The standardized raw milk was pasteurized at 72°C for 15 seconds. Subsequently, the pasteurized milk was cooled to approximately 7°C and pre-acidified (PA) at a constant rate using a food-grade lactic acid aqueous solution (88% lactic acid diluted at a 1:4 ratio, purchased from Henan Jindan Lactic Acid Technology Co., Ltd. (CAS: 50-21-5)) for 45 minutes, precisely adjusting the pH of the raw milk to 6.0.

[0063] 3. Pre-fermentation The pre-acidified raw milk was heated to 33°C. 20g of freeze-dried Streptococcus thermophilus direct starter culture (STI-13, ChrHansen) was added. After adding the starter culture, the raw milk was pre-fermented for 60 minutes.

[0064] 4. Curd Then, based on the total casein (2.5 kg), add 14.4 ml of microbial rennet (MICROLANT® Supreme 200 NB, ChrHansen) with an activity ≥190 IMCU / ml (based on 5.75 ml / kg casein), gently stir until homogeneous, and let it stand to curdle for 27 minutes. Once the curd has reached the appropriate firmness, cut it with a 2.54 cm wide knife and let it stand to heal for 15 minutes.

[0065] 5. Cooking and draining whey Subsequently, the curd particles were cooked at approximately 38°C for 20 minutes. When the pH of the curd particles dropped to approximately 5.70, whey was slowly drained, a process that lasted approximately 30 minutes. At the end of the whey drainage, the pH of the curd particles was approximately 5.50–5.65.

[0066] 6. Crushing and stretching The curd was cut into blocks and divided into two parts, and then pulverized separately when the pH value of the curd dropped to pH 5.30. The pulverized curd particles were mechanically stretched in hot water at 75°C for 8 minutes to bring the curd outlet temperature to about 60°C until a smooth, uniform cheese mass was formed.

[0067] 7. Shaping, Cooling and Salting The stretched hot cheese dough is extruded into shape (e.g., a 9×9×27 cm rectangular mold) and then immersed in a cold water bath to cool for 60 minutes, allowing the internal temperature to drop below 38°C. Afterward, the cheese blocks are placed in a 4°C, 20% (w / w) brine solution for 4 hours to salt.

[0068] 8. Second inoculation and packaging After salting and draining, before packaging, evenly spray or coat the surface of the cheese blocks with a suspension of a selected salt-tolerant flavor-enhancing starter culture (Lactococcus lactis subsp. milk fat, LL-50, DSM), controlling the inoculation concentration to 1×10⁻⁶. 6 CFU / g. The cheese cubes were then immediately sealed in high-barrier packaging bags using a vacuum packaging machine.

[0069] 9. Maturation: The product is matured at 4℃ for 20 days to obtain mozzarella cheese.

[0070] Example 2 Mozzarella cheese was prepared according to the method in Example 1, with the difference being: 1. Raw material processing: The casein content is increased to 3.5%, and the fat content is adjusted accordingly to 3.33% (maintaining the casein:fat ratio ≈ 1.05).

[0071] 2. Pre-fermentation: Increase the amount of freeze-dried Streptococcus thermophilus direct starter culture (STI-13) according to the casein content ratio. Based on a standard addition of 20g / 100kg of raw milk with a casein content of 2.5%, the total casein content in 100kg of raw milk in this example is 3.5kg, which translates to 28g of added starter culture. To balance the acid production rate and obtain similar acid production curves for raw milk with different casein contents, the pre-fermentation time is extended to 70 minutes.

[0072] 3. Curdling: The amount of rennet added is calculated based on the total amount of casein in the raw milk. In this example, the total amount of casein in 100kg of raw milk is 3.5kg. Based on the calculation of adding 5.75ml of rennet (activity ≥190 IMCU / ml) per kilogram of casein, the amount of microbial rennet added is 20.1ml.

[0073] Example 3 Mozzarella cheese was prepared according to the method in Example 1, with the difference being: 1. Raw material processing: The casein content is increased to 4.0%, and the fat content is adjusted accordingly to 3.81% (maintaining the casein:fat ratio ≈ 1.05).

[0074] 2. Pre-fermentation: Based on raw milk with a casein content of 2.5%, increase the amount of main starter culture to 32 grams, using a standard addition ratio of 20g / 100kg. To balance the acid production rate, extend the pre-fermentation time to 75 minutes.

[0075] 3. Curd: Based on the total amount of casein (4.0 kg), the amount of microbial rennet added should be increased to 23.0 ml.

[0076] Example 4 Mozzarella cheese was prepared according to the method of Example 3, except that in the crushing and stretching step, the curd was crushed and stretched at pH 5.20.

[0077] Example 5 Mozzarella cheese was prepared according to the method of Example 3, except that in the crushing and stretching step, the curd was crushed and stretched at pH 5.50.

[0078] Example 6 Mozzarella cheese was prepared according to the method of Example 3, except that in the crushing and stretching step, the curd was crushed and stretched at pH 5.00.

[0079] Example 7 Mozzarella cheese was prepared according to the method in Example 3, except that the inoculation concentration of *Lactococcus lactis* subsp. milk fat (LL-50) was increased to 5 × 10⁻⁶ in the second inoculation step. 6 CFU / g.

[0080] Example 8 Mozzarella cheese was prepared according to the method in Example 3, except that the inoculation concentration of *Lactococcus lactis* subsp. milk fat (LL-50) was reduced to 10 in the secondary inoculation step. 4 CFU / g.

[0081] Example 9 Mozzarella cheese was prepared according to the method in Example 3, except that the inoculation concentration of *Lactococcus lactis* subsp. milk fat (LL-50) was increased to 10 in the second inoculation step. 8 CFU / g.

[0082] Example 10 Mozzarella cheese was prepared according to the method of Example 3, except that in the second inoculation step, Lactococcus lactis subsp. milk fat (LL-50) was replaced with direct-inoculation cheese starter CHOOZIT™ RA21 LYD 50 DCU (Dansconis).

[0083] Example 11 This embodiment integrates all preferred parameters to demonstrate the best implementation method of this application. Mozzarella cheese is prepared according to the method of Example 1, with the following difference: 1. Raw material processing: Standardized preparation of 100 kg of raw milk with a casein content of 4.0% and a fat content of 3.81% (casein:fat ratio ≈ 1.05). Pre-acidify to pH 6.0, add 3.2 g of main starter, and pre-ferment for 75 minutes. Add 23.0 ml of rennet.

[0084] 2. Crushing and stretching: The curd blocks were crushed and stretched at pH 5.20.

[0085] 3. Secondary inoculation and packaging: The bacterial concentration is precisely controlled at 5 × 10⁻⁶. 6 CFU / g.

[0086] Comparative Example 1 The traditional method for preparing low-moisture partially defatted mozzarella cheese is as follows: The same standardized raw milk (2.5% casein, 2.38% fat) as in Example 1 was used, but without pre-acidification, and only STI-13 was used for a single fermentation without secondary inoculation. All other process parameters (such as curdling, whey drainage pH, pulverization pH, etc.) were kept as consistent as possible with those in Example 1.

[0087] Comparative Example 2 The effectiveness of the technique of "using high casein concentrate milk" was evaluated separately to exclude interference from secondary inoculation. The specific process is as follows: The same raw milk (2.5% casein, 2.38% fat) and the same process flow as in Example 1 were used, but the final secondary inoculation step was omitted.

[0088] Comparative Example 3 The effectiveness of the "second inoculation" technique in traditional raw milk was evaluated separately. The specific process is as follows: The same raw milk (2.5% casein) as in Example 1 was used, but ultrafiltration was not performed. The rest of the process was the same as in Example 1 (including pre-acidification and secondary inoculation).

[0089] Comparative Example 4 Verifying the "timing control" of the secondary inoculation is crucial, rather than simply adding bacterial strains. The specific process is as follows: The same raw milk (2.5% casein, 2.38% fat) as in Example 1 was used. The LL-50 inoculum for secondary inoculation was added together with the main starter culture STI-13 before coagulation, and the total bacterial count, after conversion, was the same as the secondary inoculation amount in Example 1. Subsequent processes remained unchanged.

[0090] Comparative Example 5 The fundamental role of pre-acidification in stabilizing concentrated milk processing and regulating calcium balance was verified. The specific process is as follows: The same raw milk (2.5% casein, 2.38% fat) as in Example 1 was used, but the pre-acidification step was omitted, and the main starter was added directly after heating. Subsequent processes (including secondary inoculation) remained unchanged.

[0091] Comparative Example 6 Compared with common flavor enhancement methods in existing technologies, this application highlights the natural advantage of its "bio-fermentation" flavor enhancement pathway. The specific process is as follows: The same raw milk and front-end process as in Example 1 were used, but without secondary inoculation. Before cheese forming, commercial cheese flavoring (imitating cream and nut flavors) was added directly, with the amount added aimed at achieving a flavor intensity similar to that of Example 1.

[0092] Test case 1. The composition of mozzarella cheese in Examples 1-11 and Comparative Examples 1-6 was determined separately, and the specific detection methods are as follows: The determination of protein, fat, moisture, calcium and dimethylglyoxal content shall be performed in accordance with GB 5009.5-2016, GB5009.6-2016, GB 5009.3-2016, GB 5009.92-2016 and GB 1886.51-2015 respectively.

[0093] The results are shown in Table 1. It can be seen that, compared to Comparative Examples 1, 2, and 4, the glycan content in the cheese prepared by the method of this application is significantly increased, and the enrichment effect of flavor compounds is more prominent. These results fully demonstrate that the secondary inoculation process and the specific inoculation timing have a significant promoting effect on the formation of key flavor compounds in cheese, and can effectively improve and enhance the overall flavor quality of cheese.

[0094] Furthermore, the comparative results of Examples 8-10 show that both the type and amount of the second fermenting agent have a significant impact on the diacetyl content in cheese. Only by selecting *Lactococcus lactis* subsp. *fatty acid bacteria* and controlling its concentration at 10... 6 ~10 7 Only within the CFU / g range can the content of dimethylglyoxal (DME) be effectively and stably increased.

[0095] Table 1: Cheese Composition

[0096] 2. Sensory evaluations were conducted on the mozzarella cheeses of Examples 1-11 and Comparative Examples 1-6, respectively. The specific method was as follows: 100g of shredded mozzarella cheese was evenly placed on a 9-inch diameter pizza base. The base was baked in a conveyor oven at 250°C for 300 seconds, cooled for 120 seconds, or until the sample temperature reached 75°C. The resulting sample was then evenly cut into 8 pieces for evaluation. Ten professional sensory evaluators were selected, and the evaluation content was as follows: Tensile properties: Take one sample and observe its tensile properties.

[0097] Melting properties: Observe the melting properties of the cheese surface under light, and pick up the pizza to observe its flowability.

[0098] Focal spot size: The size and uniformity of the focal spot are measured under light using a ruler.

[0099] Color of charred spots: Observe the color of the cheese under light.

[0100] Scorched area: Observe the scorched area of ​​the cheese under light and estimate it.

[0101] Oiliness: Press the surface of the cheese with a knife and fork or tilt the pizza under light and observe.

[0102] Taste and smell: Take one sample, smell it first, then rinse your mouth with warm water, and then taste it.

[0103] Texture: Take one sample, rinse your mouth with warm water, and then taste it to experience its texture.

[0104] The taste and aroma rating scale is shown in Table 2.

[0105] Table 2: Taste and Aroma Rating Table

[0106] The results are shown in Table 3. It can be seen that the mozzarella cheese prepared in Examples 1-11 has relatively better melting properties, stringiness, and flavor compared to Comparative Examples 1-6.

[0107] The increased casein content in the raw milk of Examples 2 and 3 resulted in a denser protein network in the cheese compared to Example 1. After maturation and baking under the same conditions, the cheese was expected to exhibit superior firmness, elasticity, and stringiness, demonstrating the positive impact of high-casein raw materials on improving texture and pizza functionality.

[0108] In Example 4, the curd was pulverized at a lower pH (pH 5.20), which further optimized the calcium cross-linking state of the casein network, resulting in a more uniform and oriented fiber structure. Compared to Example 3 (pulverized at pH 5.30), the resulting cheese had a longer string length of 35-40 cm and better toughness. It melted more evenly and smoothly upon heating, achieving a better balance between hardness and elasticity, and a more tender and chewy texture, further improving its overall baking performance. When the curd was pulverized at a slightly higher pH (pH 5.50), as in Example 5, its texture became softer, its elasticity decreased, and it might collapse after melting due to excessive softness, resulting in poor stringing properties. When the curd was pulverized at an excessively low pH (pH 5.00), it caused excessive dissolution of colloidal calcium phosphate, leading to the loss of necessary calcium cross-linking support in the casein micelles and a weakened protein network structure. The resulting cheese was difficult to form a continuous fibrous structure during heat stretching, resulting in a significant decrease in its stringing properties (length <15cm and easily broken), a loose texture, and even a powdery feel. Increased free oil precipitation during heating and uneven formation of charred spots further contributed to its significantly inferior overall functional properties compared to Example 3. This demonstrates the importance of the preferred low grinding pH range for obtaining optimal functional texture in this application.

[0109] Example 7: The concentration of the second inoculation was increased to 5 × 10⁻⁶. 6 CFU / g, compared to an inoculation concentration of 1×10 6 Example 3, with CFU / g, yielded cheese that, according to sensory evaluation and diacetone content testing, exhibited significantly enhanced flavor intensity and complexity, fully demonstrating the significant advantages of the optimized inoculation concentration in targeted and efficient enhancement of cheese flavor; when the secondary inoculation concentration was reduced to 10... 4 At CFU / g, the flavor enhancement effect on the cheese was weak compared to Examples 3 and 7; however, when the concentration for the second inoculation was further increased to 10... 8 At CFU / g, compared to Examples 3 and 7, the flavor of the cheese showed a deteriorating trend (e.g., off-flavors, flavor imbalance, etc.). Therefore, the 10% concentration defined in this application... 6 ~107 The CFU / g secondary inoculation concentration range is a necessary condition for achieving significant improvement in cheese flavor.

[0110] In Example 10, the secondary inoculation of *Lactococcus lactis* subsp. *milk fat* (LL-50) was replaced with CHOOZIT™ RA21 LYD50 DCU. The resulting cheese showed significantly deteriorated flavor intensity, complexity, and typicality compared to Example 3. This study indicates that different strains exhibit varying flavor metabolism during cheese maturation. While both *Lactococcus lactis* subsp. *lactolaccos* and *milk fat* subsp. *lactolaccos* are commonly used in dairy fermentation, the contribution of specific strains to characteristic flavors is irreplaceable. The cheese obtained in this example exhibited a bland flavor, lacking the rich creamy aroma and layered characteristics characteristic of the LL-50 strain. This suggests that not all conventional flavor bacteria are suitable for secondary inoculation, and that the LL-50 strain selected in this application demonstrates a specific synergistic compatibility with the secondary inoculation process.

[0111] Example 11 integrates the superior functionality of a high-casein matrix, texture optimization from low-pH grinding, and highly efficient flavor-targeted inoculation. The product is expected to achieve optimal pizza baking performance (extra-long cheese pull, uniform melting and browning) after maturation at 4°C for 15-20 days, while also exhibiting an extremely rich and full-bodied creamy and nutty post-ripening flavor, with highly consistent quality between batches.

[0112] Comparative Example 1 uses the most traditional low-moisture partially defatted mozzarella cheese preparation process. Cheese products prepared using this process have a single flavor and may have a hard texture or poor melting properties due to different calcium balance.

[0113] Comparative Example 2 used the same raw milk (2.5% casein) and the same process as Example 1, but omitted the secondary inoculation step. Compared with Example 1, the cheese produced in this example had significantly insufficient flavor and a bland, thin texture. Although using high casein concentrate, this comparative example was comparable to Example 1 in terms of texture and functionality (such as stringiness and melting properties). However, due to the lack of post-fermentation resulting from the secondary inoculation, the content of key flavor compounds such as diacetyl was significantly lower than that of Example 1. In sensory evaluation, it only had a basic milky aroma and lacked the rich creamy flavor and complexity of Example 1. Therefore, this comparative example demonstrates that, based on the high casein concentrate process, secondary inoculation is the only variable that brings about a significant flavor improvement, thus highlighting its independent technical contribution.

[0114] Comparative Example 3 used the same raw milk (2.5% casein) as Example 1, but without ultrafiltration. The remaining processes were the same as in Example 1 (including pre-acidification and secondary inoculation). Compared to Example 1, the cheese produced had significantly insufficient functionality, specifically exhibiting shorter string length (approximately 15-20 cm), poor melting properties (uneven melting), and a softer texture lacking elasticity. This demonstrates that even with secondary inoculation, without combining it with high-casein concentrate to optimize the matrix (i.e., without ultrafiltration), the product's functionality cannot reach its optimal level, proving the synergistic necessity of the two technologies.

[0115] Comparative Example 4 added the LL-50 microbial agent, which was inoculated twice, together with the main fermenting agent STI-13 before curdling. Compared with Example 1, the flavor of the cheese prepared by this method was significantly deteriorated (the flavor bacteria could not play an effective role in the acidic and competitive environment in the early stage). This proves that the flavor fermenting agent should be inoculated after dry salting to effectively improve the flavor of the cheese.

[0116] In Comparative Example 5, no pre-acidification treatment was used. Compared with Example 1, the cheese prepared in this example had poorer curdling characteristics, whey drainage efficiency, and uniformity of final texture due to different initial calcium balance. This proves that pre-acidification is an important basis for stabilizing the early process and ensuring the repeatability of subsequent steps in the method of this application.

[0117] In Comparative Example 6, additives were used to replace secondary inoculation. Compared with the example, the cheese prepared by this method may have a similar basic flavor, but it lacks the complex layers of natural fermentation flavor and post-ripening changes. In addition, the product label must indicate "edible flavoring", which does not conform to the trend of clean labeling. This highlights the advanced nature and market value of the technical route of this application.

[0118] Table 3: Sensory evaluation of mozzarella cheese in the examples and comparative examples

[0119]

[0120]

[0121] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A process for the preparation of a mozzarella cheese, characterized by, The method comprises the following steps: defatting and ultrafiltrating whole milk to obtain raw milk; sterilizing and pre-acidifying the raw milk to obtain acidified liquid; inoculating the acidified liquid with a first starter culture and performing pre-fermentation to obtain a pre-fermentation product; sequentially performing curd formation, whey separation, crushing, hot stretching, cooling and salting on the pre-fermentation product to obtain a salting product; inoculating the surface of the salting product with a second starter culture and performing ripening to obtain the mozzarella cheese; wherein the first starter culture is selected from Streptococcus thermophilus; The second starter culture is selected from *Lactococcus lactis* subsp. *milk fat*, and the concentration of the second starter culture on the surface of the salted product is 10. 6 ~10 7 CFU / g.

2. The method of claim 1, wherein, The method satisfies at least one of the following conditions: The mass fraction of casein in the raw milk is 2.5%-4.0%; The mass ratio of casein to fat in the raw milk is 1:(1-1.1); The mass fraction of protein in the raw milk is 5%-6%.

3. The method of claim 1, wherein, The method satisfies at least one of the following conditions: The sterilization temperature is 72-74°C, and the sterilization time is 14-16 s; The pre-acidification is performed using an acidifying agent, and the acidifying agent comprises one or more of lactic acid, citric acid, acetic acid, malic acid, hydrochloric acid, phosphoric acid or gluconic acid-delta-lactone; The pre-acidification time is 40-50 min; The pH of the acidified liquid is 5.5-6.

5.

4. The method of claim 1, wherein, The method satisfies at least one of the following conditions: The mass fraction of casein in the raw milk is 2.5%, and the first starter culture is added in an amount of 15-25 g based on 100 kg of the acidified liquid; When the mass fraction of casein in the raw milk is less than 3.5%, the pre-fermentation time is 50-70 min; and when the mass fraction of casein in the raw milk is not less than 3.5%, the pre-fermentation time is 70-80 min; The pre-fermentation temperature is 33-37°C.

5. The method of claim 1, wherein, The curd formation comprises adding rennet to the pre-fermentation product, stirring and then standing, and cutting into blocks; The whey separation comprises heating the curd formation product, and when the pH of the heated product is 5.6-5.7, separating the whey.

6. The method of claim 5, wherein, The method satisfies at least one of the following conditions: The mass-volume ratio of casein to rennet is 1 kg:(5-6) mL, calculated based on the total content of casein in the raw milk; The enzyme activity of the rennet is ≥190 IMCU / ml; The standing time is 25-35 min; After cutting into blocks, standing for 10-20 min; The heating temperature is 35-40°C; The heating time is 15-25 min; The whey separation time is 25-35 min; The pH of the curd after whey separation is 5.50-5.

65.

7. The method of claim 1, wherein, The crushing comprises crushing the curd after whey separation, which has a pH of 5.10-5.

40.

8. The method of claim 1, wherein, The hot stretching comprises stretching the curd obtained by crushing in hot water at 70-80°C for 5-10 min; The cooling treatment comprises placing the product after the hot-dip stretching treatment in a cold water bath for 50-70 min until the internal temperature of the product is below 38℃; The salt treatment comprises placing the product after the cooling treatment in a 18-22% salt solution for 3-5 h; The temperature of the ripening treatment is 2-6℃ and the time is 15-20 days.

9. A mozzarella cheese, characterized in that, The mozzarella cheese is obtained by the method of any one of claims 1-8.

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