{"paper_id":"45517d63-84d1-401e-b60c-bbd3bfe4bc82","body_text":"Effect of pre-acidification and whey proteins on low-fat Mozzarella cheese during storage: physicochemical, textural, and functional properties | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of pre-acidification and whey proteins on low-fat Mozzarella cheese during storage: physicochemical, textural, and functional properties Deju Zhang, Juan Xiong, Kai Jiang, Xiaorui Zhao, Yiming Gan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4055582/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract The aim of the current study was to explore the effect of pre-acidification and whey protein-based fat replacers (CH-4560 and YO-8075) on low-fat Mozzarella cheese (LFC). The color and browning, calcium, meltability, texutre and pizza bake performance of cheeses at 0, 7, 14, 21, 28 days storage was determined, and full-fat cheeses (FFC; milk containing 2.21 g fat/100 g milk) and LFC were manufactured as control. The LFC without fat replacer was harder, less sticky, and had high stretch force and low meltability, as well as poor pizza bake and stretch performance, while these appearance defects were partially corrected via the addition of whey protein concentrates and using pre-acidification. Pre-acidification of cheese milk to pH 5.5 or lower negatively affected the stickiness, and pizza bake, and stretch performance of LFC and LFC with fat replacers. As aging progressed, the meltability, stickiness and stretch performance of Mozzarella cheese improved, whereas the hardness and stretch force of Mozzarella cheese decreased. Our study will provide guidance for the preparation of higher quality low-fat mozzarella cheese. Mozzarella cheese Pre-acidification whey proteins low-fat texture Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Fats have many functional properties in a food carrier as they play a significant role in the texture, color, and sensory properties of foods in many specific ways [1]. However, excessive consumption of fat is related to a lot of health risks, such as obesity, atherosclerosis, hypertension, diabetes, inflammation, gall bladder disease, and cancer [2]; this has aroused the concern of consumers that dietary fat intake should be tightly controlled to reduce the risk of multiple diseases [3]. Driven by the expansion of the pizza industry, Mozzarella cheese sales achieve substantial growth and currently become the most commonly purchased cheese in the US [4]. Full-fat Mozzarella cheese (FF) is conventionally used as a topping on pizza, because of its good melting and stretching properties [3]. But they may contain more than 20% fat, which indicates a health threat to cheese lovers [5]. In view of the current situation, many studies were conducted to study the functionality of low-fat Mozzarella cheese (LF). The reduction of fat in LF could cause a reduction in oiling off and moisture to protein values, which gives the cheeses poor functionality and limits their use on pizza [6]. Moreover, fat-reduced cheese is conventionally low in whiteness values and may exhibit an unappealing appearance [5]. To overcome these drawbacks, several studies were carried out to improve the quality of LF, such as the use of pre-acidification, EPS-producing culture, and fat replacer [3,7]. Among them, whey protein concentrates are commonly used due to their high nutritional value and good functionality, such as foaming, gelling and emulsifying properties [8], which allows them to be used as fat replacers in many food systems [9]. Low-fat dairy products with whey protein may exhibit comparable functional properties to their full-fat counterparts. Therefore, determining the functionality of low-fat cheeses containing whey protein concentrates (WPC) is beneficial for promoting the quality of LF and providing a reference for the utilization of whey protein in the cheese industry. Pre-acidification also plays an important role in Mozzarella cheese functionality and texture by affecting the cross-linking between calcium and protein [10]. High levels of bound calcium in LF are positively related to denser cheese structure and higher values of hardness [6]. Pre-acidification treatment could remove bound calcium from casein micelles and reduce the cross-linking of casein, substantially producing a low-fat cheese with improved qualities [10]. However, over-acidification results in excessive bound calcium being released from the curd, thus yielding cheese with excessive softness and high flowability. It is also not acceptable for Mozzarella cheese to be used as a pizza topping. Thus, the aim of this study was to investigate the effect of combining the technique of pre-acidification and the use of whey protein concentrates on hardness, melt, color, stretch properties and pizza bake properties of Mozzarella cheese. As a result, the best final pH of pre-acidification was selected for follow-up studies. Specifically, FF, LF, and LF supplemented with whey protein concentrate (LFWP) were determined for evaluating the potential of LFWP to be used in pizza industry. 2. Materials and Methods 2.1 Materials Pasteurized and homogenized milk (full-fat milk: fat content: 5.1 g/100 mL, low-fat milk: fat content: 2.5 g/100 mL, and skimmed milk: fat content: 0.7 g/100 mL) were purchased from local distributors (Farm Milk Company Limited, Hong Kong). Both the Nutrilac® CH-4560 and Nutrilac® YO-8075 were commercial whey protein concentrates and were supplied by Arla Food Amba (Copenhagen, Denmark). Double-strength chymosin was purchased from Chr. Hansen, Bayswater, Victoria, Australia. Streptococcus thermophilus ASCC 1275 (ST) , Lactobacillus delbrueckii ssp . bulgaricus ASCC 859 (LB), Lactobacillus casei ASCC 290 (LC), and Lactobacillus paracasei ASCC 276 (LP) were obtained from the Australian Starter Culture Research Center (ASCRC, Werribee, Australia). 2.2 Culture propagation All microorganisms were stored in sterile MRS broth with 50 g/L glycerol at -80 ℃. To activate the ST, LB, LC, and LP cultures, a 100 μL aliquot of each culture was individually transferred into MRS broth and incubated in a CO 2 incubator (Shel-Lab Co., Cornelius, U.S.A.) at 37 ℃ for 18 h following the method of Zhang , et al. [11]. After the second transfer in MRS broth, the activated starter cultures were transferred into 10% (w/w) reconstituted skim milk for another two transfers. Activated cultures were used for cheese making. 2.3 Cheese making Mozzarella cheeses were made following the method of Ayyash and Shah [12] with some modifications. An aliquot of 2 L fresh milk was cultured with 1 mL/100 mL inoculum of each bacterial strain and acidified with 100 mM citric acid to pH 5.9, 5.8, 5.7, 5.6, and 5.5 at 37 ℃ in a custom-made, temperature-controlled cheese vats. Eighteen batches of Mozzarella cheeses were manufactured as indicated below. Table 1 Mozzarella cheeses made from milk pre-acidified to different pH No. Cheese Fat content (g/100 g milk) Preacidification pH Fat replacer 1 FF9 2.21 5.9 / 2 LF9 1.10 5.9 / 3 LFC9 1.10 5.9 CH-4560 4 LFY9 1.10 5.9 YO-8075 5 FF8 2.21 5.8 / 6 LF8 1.10 5.8 / 7 LFC8 1.10 5.8 CH-4560 8 LFY8 1.10 5.8 YO-8075 9 FF7 2.21 5.7 / 10 LF7 1.10 5.7 / 11 LFC7 1.10 5.7 CH-4560 12 LFY7 1.10 5.7 YO-8075 13 LF6 1.10 5.6 / 14 LFC6 1.10 5.6 CH-4560 15 LFY6 1.10 5.6 YO-8075 16 LF5 1.10 5.5 / 17 LFC5 1.10 5.5 CH-4560 18 LFY5 1.10 5.5 YO-8075 The curd made from high-fat milk (fat content: 2.21 g/100 g) pre-acidified to pH 5.6 or 5.5 was too thin and can not be piled into slabs. Streptococcus thermophilus ASCC 1275 , Lactobacillus delbrueckii ssp . bulgaricus ASCC 859, Lactobacillus casei ASCC 290, and Lactobacillus paracasei ASCC 276 were used in all treatments. Nutrilac® CH-4560 and Nutrilac® YO-8075 (10 g) were added directly to cheese milk and mixed for 1 min. After 40 min of aging, 1 mL chymosin diluted in 19 mL distilled water was added to the cheese milk, followed by a brief stirring. The milk got coagulated after 45 min and then it was cut into 1 cm 3 cube with a knife. The cut curd was cooked as described by Dai , et al. [13] at 37 ℃ until the pH of the curd dropped to 5.3. During this time, whey was drained off and the cooked curd was piled and chopped into slabs. Thereafter, a salting process (1.5 g/100 g) followed and curds were mellowed for another 20 min. All cheeses were hand-stretched in hot brine solution (4 g/100 g) maintained at 75 ℃ and then kneaded in blocks. After stretching and kneading, the 200 g blocks of Mozzarella cheese were vacuum packaged and aged at 4 ℃ for 2 weeks. Sampling was carried out to determination of functionality and chemical composition on days 0, 7, and 14 of storage. 2.4 Composition Moisture was determined by a moisture analyzer (HB43-S, Mettler Toledo, Switzerland), protein by Kjeldahl method, and fat by Babcock method as described by Dai, Jiang, Corke and Shah [13] and Liu , et al. [14]. The cheese yield was calculated as the weight of total cheese to the weight of total materials, multiplied by 100. All analyses were performed in triplicate. 2.5 Color The color parameters of cheeses were analyzed with a Minolta Chroma Meter (CR-300 Series, Minolta, Japan) following the method of Zhao , et al. [15]. The surface color of cheeses was taken at 4 external and 4 internal locations of the cheeses on days 0, 7, and 14 of storage. An aliquot of the ground cheese (15 g) was weighed into a disk (7.5 cm × 7.5 cm × 1.5 cm) and was heated at 100 ℃ for 160 min. After cooling, the color of cheeses was measured again, this represented the browning of cheeses. The color and browning of cheese were expressed as L* (0-100, black to white), a* (green (-) to red (+)), and b* ( blue (-) to yellow (+)). 2.6 Calcium Cheese (0.4 g) with 2 mL of 30 % w/w H 2 O 2 and 6 mL of nitric acid was digested using a Microwave Digester (ETHOS-1600, Milestone, Italy) for 90 min. Afterward, the calcium content of cheese was analyzed using an atomic absorption spectrophotometer (iCE 3300, Thermo Scientific, UK) with an air (13.5 L/min)/acetylene (2.0 L/min) flame as indicated by Zisu and Shah [10]. The calcium atomic spectroscopy standard containing 993 μg/mL calcium (Sigma‐Aldrich, St. Louis, MO, USA) was applied to prepare working standards in a sample buffer (49.2% deionized water, 48.7% of 12% (w/v) trichloro-acetic acid and 2.1% of 5% (w/v) lanthanum oxide). 2.7 Meltability The meltability of cheeses was determined according to the method of Cais-Sokolinska , et al. [16] with some modifications. Grated cheese (10 g) was placed in a glass tube (30 mm × 200 mm) and packed to form a plug at the bottom. The cheeses were heated at 100 ℃ for 12 min and the length of the cheese was measured before heating and after the cheeses were cooled down to room temperature. The meltability of cheese was expressed as the length of cheese after heating minus the length of cheese before heating and the percentage meltability was calculated as length difference divided by the initial length of cheese and multiplied by 100%. 2.8 Pizza bake Pizza dough was manually made and baked as described by Zhang, Ling, Jiang, Zhao and Gan [7]. Then cheese (20 g) was cut into shreds (approximetely 2 mm × 2 mm × 20 mm pieces) using a cheese grater and spread on the top of the pizza base, followed by baking at 220 ℃ for 5 min. The changes in Mozzarella cheese were observed visually for shred fusion, melting and scorching degree, and extent of blistering. 2.9 Texture The hardness and stickiness of Mozzarella cheeses were determined using a TA.XT Plus model texture analyzer (Stable Micro Systems, Godalming, Surrey, UK) following the method of Liu , et al. [17] and Zhang , et al. [18] with modifications. The Mozzarella cheeses were cut into cubes (30 mm × 30 mm × 20 mm) and placed on the stage of the texture analyzer. The TPA conditions were set as follows: test speed of 3.0 mm/s, and test distance of 20 mm. The stretch force and stretch performance of the melted cheese were also determined following the method of Dai, Jiang, Corke and Shah [13]. Cheese samples were cut into small cubes and filled evenly in a plastic pot with a fork. Then the sample pot with fork and cheese cubes was heated in a microwave oven (600 W) for 40 s and tested immediately after the temperature of the melted cheese reached 55 ℃. The TPA conditions were set as follows: test speed of 20 mm/s and test distance of 270 mm. The stretch performance of Mozzarella cheeses was recorded using a camera. 2.10 Statistical data evaluation For each assay, at least three independent samples were prepared. Results were given as mean ± standard deviation (SD) (n = 3). Figures in this chapter were drawn with GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). 3 Results and discussion 3.1 Composition Table 2 shows the composition of FF and LF. The protein and moisture content of FF were at low level (protein content was 16.44%, 16.06%, and 15.37%, and moisture content at D 0 was 58.07%, 59.93%, and 60.93% for FF9, FF8, and FF7, respectively) compared to LF and LFWP, and it was expected due to the hydrophobicity of fat globules. As the pre-acidified pH of cheese milk decreased, the yield and moisture content of Mozzarella cheese increased, while the fat and protein content decreased. This is perhaps because the removal of bound calcium from casein micelles caused by pre-acidification increased the hydration of the cheese para-casein matrix, thereby increasing the moisture content of cheese [19,20]. The moisture and yield of LFWP were higher than that of the LF, corresponding to the fact that whey protein has excellent moisture retention capacity. Finally, we also found that the moisture of cheese showed fluctuation in the range of 58.22% to 65.23% and remained stable over storage time. Table 2 Yield and composition of Mozzarella cheese Cheese Yield (%) Fat (%) Protein (%) Moisture (%, D 0) Moisture (%, D 7) Moisture (%, D 14) FF 9 13.17 ± 0.51 15.6 ± 0.26 16.44 ± 1.15 58.07 ± 1.51 58.02 ± 0.53 58.22 ± 1.56 LF9 10.50 ± 0.15 10.63 ± 0.85 22.11 ± 1.19 59.04 ± 0.95 57.94 ± 0.52 58.47 ± 1.43 LFC9 10.76 ± 0.48 9.10 ± 0.56 22.51 ± 1.56 60.53 ± 1.88 58.98 ± 1.28 58.67 ± 1.56 LFY9 11.77 ± 0.23 9.17 ± 0.35 21.88 ± 0.67 60.99 ± 0.82 59.09 ± 0.94 58.82 ± 1.04 FF8 12.26 ± 0.35 15.90 ± 0.14 16.06 ± 0.69 59.93 ± 1.03 60.14 ± 1.49 59.13 ± 1.16 LF8 10.20 ± 0.46 8.50 ± 0.28 22.26 ± 0.16 60.54 ± 1.00 59.66 ± 1.06 59.67 ± 1.17 LFC8 11.25 ± 0.48 8.87 ± 0.42 21.85 ± 0.88 61.41 ± 0.83 60.90 ± 0.78 60.20 ± 0.65 LFY8 11.98 ± 0.22 8.83 ± 0.61 21.75 ± 1.08 61.24 ± 0.62 60.73 ± 0.89 60.18 ± 0.48 FF7 11.93 ± 0.31 15.10 ± 0.40 15.37 ± 0.18 60.93 ± 0.48 60.54 ± 0.71 59.96 ± 0.46 LF7 10.75 ± 0.26 8.30 ± 0.80 20.17 ± 0.67 63.72 ± 0.32 62.32 ± 1.81 61.53 ± 2.37 LFC7 10.29 ± 0.90 7.80 ± 0.85 20.20 ± 1.21 64.34 ± 0.70 64.15 ± 1.03 63.74 ± 0.70 LFY7 10.95 ± 0.13 7.95 ± 0.35 19.88 ± 0.70 64.46 ± 0.83 63.70 ± 1.14 63.66 ± 0.89 LF6 10.58 ± 1.37 7.93 ± 0.12 20.29 ± 0.11 62.00 ± 1.96 63.00 ± 1.77 62.03 ± 1.85 LFC6 11.88 ± 0.39 7.37 ± 1.03 19.55 ± 0.62 65.00 ± 2.02 64.35 ± 1.28 63.36 ± 1.22 LFY6 11.51 ± 0.27 7.30 ± 0.36 19.66 ± 1.59 65.18 ± 0.52 64.19 ± 1.02 63.14 ± 1.29 LF5 12.10 ± 0.41 7.65 ± 0.78 19.39 ± 0.83 64.35 ± 0.77 65.04 ± 1.68 64.11 ± 1.97 LFC5 12.95 ± 0.07 7.25 ± 0.64 18.76 ± 0.90 65.5 ± 1.63 65.69 ± 0.87 65.23 ± 0.47 LFY5 12.73 ± 0.37 7.20 ± 0.60 18.78 ± 0.98 65.34 ± 1.38 65.40 ± 1.07 65.01 ± 0.44 Values are expressed as mean ± standard deviation. 3.2 Color and browning The color and browning values of Mozzarella cheeses during storage are given in Figure 4.1. The browning L* values of Mozzarella cheese were lower compared to the color L* values of Mozzarella cheeses. Besides, it was found that the color and browning values of FF were higher than those of LF and LFWP during the entire period of storage. This difference can be interpreted as fat globules contribute to the lightness of cheeses by scattering light [21]. As expected, significant differences were found in color and browning values between LF and LFWP, showing that the addition of whey protein in Mozzarella cheeses improved the whiteness of Mozzarella cheese before and after baking. The improved color L* values in LFWP might be attributed to more light penetrating the superficial layers is scattered by the edges of the whey pockets [22]. The higher browning L* values of LFWP were possibly due to free water in cheese that can protect the casein matrix from dehydration during baking by adsorbing heat energy. In addition, results also showed that a decrease in the final pH of pre-acidified cheese milk induced an increase in the color and browning values of cheeses, so in the LFC5 and LFY5, these two parameters were close to those of the FF9. We also observed a reduction of color L* values and an increase of browning L* values as cheese age increased. The former was because of serum phase and proteolytic changes during storage [13], and the latter was because the active metabolism of starter bacteria reduced the reducing sugar content, further decreasing the formation of Maillard reactions products that might affect the whiteness of Mozzarella cheese. 3.3 Calcium Figure 4.2 revealed progressively declining values of the calcium content of Mozzarella cheese in response to increasing amount of citric acid added to cheese milk. Solubilization of casein micelles resulting from acidification leads to dissociation of colloidal calcium phosphate, and this solubilization will lead to the release of calcium in whey, which explains why a decreased trend in calcium content was observed as the final pH of pre-acidification of cheese milk decreased [23]. The calcium content of LF and LFWP was higher compared to FF. As suggested by Hindmarsh and Watkinson [24] and Dalgleish [25], the subunits of casein micelle are bound together through calcium phosphate bridges via van der waals forces, hydrophobic bonds, and ionic calcium linkages, which contribute to enhanced calcium content in casein matrix. Therefore, the higher calcium content in LF and LFWP might result from their higher protein content. No significant difference was observed in calcium content between LF and LFWP. Our results were in agreement with the conclusions of Zisu and Shah [10], who also saw a decrease in calcium content when cheese milk was pre-acidified with citric acid. However, there is a great difference in calcium content compared to the current investigation. This may be because of the difference in protein content between their cheeses and ours. 3.4 Meltability The observed changes in melt length and meltability during storage are shown in Figure 4.3. The average melt length of FF9, FF8, and FF7 at D 0 was 4.25 cm, 6.88 cm, and 12.30 cm compared to 0.78 cm, 2.16 cm, 5.22 cm, 7.32 cm, and 11.47 cm for LF9, LF8, LF7, LF6, and LF5, respectively. Similar results were also observed for the meltability values of Mozzarella cheeses. As the proportion of protein increases, the calcium phosphate para-casein network becomes more fused and contiguous, resulting in the displacement of adjoining planes. Thereafter, the mobility of the cheese matrix became more difficult especially as the level of free oil decreases simultaneously [5]. In addition, the higher calcium content in LF also has a negative impact on their melt length and meltability [19]. Due to these main reasons, the melt length and meltability of Mozzarella cheeses were greatly reduced in LF and LFWP. It was observed that the melt length and meltability of Mozzarella cheese enhanced as the final pH of pre-acidification decreased. There are two main reasons for this. Pre-acidification increased the moisture content of the protein matrix, which further attenuates protein-protein interactions thus plasticizing the matrix, resulting in higher mobility [26]. Secondly, the removal of calcium resulting from pre-acidification causes increased interaction of proteins with surrounding serum, resulting in higher levels of protein hydration [20]. A direct relationship between calcium content, meltability and texture of cheese has also previously been described by Joshi, Muthukumarappan and Dave [20] and Fife , et al. [27]. Cheeses containing high calcium content were surrounded by more free serums, while cheeses with low calcium content have less as these free serums are partially absorbed by the protein matrix. When free serums were absorbed by proteins, the proteins become more hydrated, which gives the protein matrix higher flowability during heating. The influence of WPC on the meltability of cheeses was also studied in this exploration. The addition of whey protein to cheese milk increased the melt length and meltability of Mozzarella cheese, and the effect of whey proteins of CH-4560 and YO-8075 treatments on Mozzarella cheese was similar. In the study of Ismail , et al. [28], they attributed this to high moisture retention of Mozzarella cheeses caused by WPCs. However, we also hold that WPCs serve as fat replacers and lubricate the casein matrix. And it could also mimic the emulsified fat via forming a dispersed phase of particles that are free to move independently [29]. These action ways allow Mozzarella cheeses containing whey protein have better melting performance. The melt length and meltability of Mozzarella cheeses increased during cold storage, and these two parameters exceeded the limit of the test (the maximum melting length is 14.5 cm) in LFC6, LFY6, and LF5 at D 7 and D 14, and in LFC5 and LFY5 at D 0, D 7, and D 14. The above trends also concur with those of others [5,27], who also found that a period of storage increased the melt length and meltability of Mozzarella cheese. The greater melting performance might be due to degradation of the casein matrix [5]. 3.5 Pizza bake The pizza bake images of Mozzarella cheeses are shown in Figure 4.4 and Figure 4.5. After baking, FF9, LF9, LFC9, LFY9, LF8, LFC8, LFY8, LF7, LFC7, and LFY7 showed incomplete shred fusion with low mobility at D 0, which is because of the relatively high final pH of pre-acidification and the lower fat content in cheeses. However, FF8, FF7, LF6, LFC6, LFY6, LF5, LFC5, and LFY5 exhibited a much better ductility and meltability, showing that pre-acidification played a significant role in the pizza bake performance of Mozzarella cheeses. It was found that LF9 exhibited scorch formation and created an unappealing surface at D 14, but the scorching decreased with the introduction of WPCs. By comparison, we can see that the LFC8 and LFY8 showed better pizza bake performance with improved melting and ductility compared to that of the FF8 and LF8 at D 0 and D 14. Similar conclusions can also be drawn by comparing the LF7 with LFC7 and LFY7, and LF6 with LFC6 and LFY6. Additionally, when the pre-acidification pH of cheese milk was no greater than 5.7, Mozzarella cheese with added whey protein demonstrated comparable baking properties to full-fat cheeses. All the Mozzarella cheeses made from cheese milk pre-acidified to pH 5.5 exhibited high meltability and flowability. However, these Mozzarella cheeses had an excessively thin melting texture, which may cause poor taste and greatly affect consumer preference. At D 14, all the Mozzarella cheese showed more complete shred fusion than at D 0. This observation was similar with the changing pattern of meltability of Mozzarella cheeses. Dai, Jiang, Shah and Corke [5] also observed incompletely shred fusion and some scorched shreds around the edge of the pizza in LFC at D 7, and these cheeses exhibited improved melting performance and less scorching after 28 days of storage. The author also reported that low-fat Mozzarella cheese showed big blister after baking at D 28, but in this study, we did not observe this phenomenon in these Mozzarella cheeses. This may be because our cheese sticks before baking were very thin, which avoided the formation of blisters in the cheese. 3.6 Texture Texture values are an important indicator of the textural characteristics of a cheese that correlates well with sensory scores [30]. Mozzarella cheese made with high-fat milk was softer than cheese made with low-fat milk (Figure 4.6). This is because the greater fat content in cheese, the more interruptions there are in the casein matrix and increased interference in the long-range interactions between the proteins. Therefore, the reduction of fat in cheese matrix allows protein interactions to be very extensive, leading to a rubbery texture [3]. The addition of whey protein concentrates contributes to the reduction of hardness of Mozzarella cheeses. As discussed previously, the whey protein concentrates could retain a certain amount of water and be inserted inertly into the pores of the protein matrix like fat globules, which may disrupt the homogeneity of the protein network and further decrease the hardness of cheese [31]. Moreover, our results showed the hardness of cheeses decreased with the increasing amount of citric acid added to cheese milk. Specifically, at D 0, the hardness values of LF9, LFC9, and LFY9 were 778.85 g, 594.14 g, and 614.01 g, respectively, while these cheeses were associated with lower hardness values when made using cheese milk pre-acidified to pH 5.5 and their hardness values decreased to 141.50 g, 108.47 g, and 111.76 g for LF5, LFC5, and LFY5. After 14 days of storage, a slight decrease in the hardness of all the Mozzarella cheeses was observed, except for FF. There was a significant difference between the Mozzarella cheeses for the parameter of stickiness (P < 0.05). As indicated in Figure 4.7, FF possessed the highest stickiness among cheeses (absolute value, similarly hereinafter), while LF had the least hardness. The interactions between fat globules and casein increase the degree of adherence between cheeses and spherical probe with which it is in contact [32]. Furthermore, the incorporation of whey protein concentrates in cheese milk increased the stickiness of Mozzarella cheese. In our study, whey proteins of CH-4560 and YO-8075 were micro-particulated whey proteins. As suggested by Fang , et al. [33], denatured whey protein concentrates could form a high molecular weight whey protein polymer similar to hydrocolloids, thus creating high level of cross-linking within the gel network, ultimately increasing the stickiness of dairy products. Higher stickiness values were noted as the final pre-acidification pH of Mozzarella cheeses decreased. This can be explained by the protein structure of the casein network. In terms of FF9, LF9, LFC9, and LFY9, they have highly aggregated protein network and there is more moisture present in serum channels. Therefore, the proteins tend to maintain their self-association and exhibited low adhesiveness. However, when a significant amount of calcium was removed from Mozzarella cheese due to pre-acidification of cheese milk to pH 5.5, the proteins are more unfolded, which allows them to interact with other surfaces. In our study, LF5, LFC5, and LFY5 had highly hydrated protein matrices and the cheese was very sticky before and after heating. Therefore, we can speculate that the unfolding of the protein aggregates in the low calcium conditions exposes more hydrophobic sites and charged sites, which gives Mozzarella cheeses a higher level of flexibility and allows the proteins to readily interact with the surface with which it is in contact. Furthermore, the stickiness of LF and LFWP declined with increasing storage time, but this downward trend did not constitute statistical significance (P > 0.05). The stretch force of Mozzarella cheeses with different final pre-acidification pH is shown in Figure 4.8. As the fat content of cheese decreased, the stretch force increased. The addition of whey protein concentrates resulted in a lower stretch force, this is especially evident in the Mozzarella cheeses made from milk pre-acidified to pH 5.9, 5.6, and 5.5. The results also showed that treatment with citric acid decreased the stretch force of Mozzarella cheeses, and the stretch force of melted cheese gradually decreased with the increasing amount of citric acid. Finally, aging also had a positive effect on the stretch force values of LF and LFWP, whereas FF9, FF8, and FF7 were not significantly affected by cold storage (P > 0.05). The stretch performance of FF, LF, and LFWP was also recorded and presented in Figure 4.9 and Figure 4.10. It is obvious that the Mozzarella cheeses made from milk pre-acidified to pH 5.6 and 5.5 had high moisture retention and exhibited smooth texture. These findings were consistent with the moisture content and the stretch force results. In terms of LF9, LFC9, LFY9, LF8, LFC8, LFY8, and LF7, the strands of these cheeses showed a rough texture. Combining the results here with the stretch force values, it can be concluded that the protein matrix of cheeses made from milk pre-acidified to high pH was tightly associated with each other, the bonds between proteins are stronger and require more energy to break. From these images, the Mozzarella cheese with WPCs had improved stretch performance with longer and less fractured strands were observed in LFWP than in LF, which may be attributed to the excellent thickening properties of whey proteins. With the increasing storage time, the Mozzarella cheeses exhibited more appealing stretch performance at d 28 as they showed longer and thicker strand formation compared to Mozzarella cheeses at d 0. This phenomenon was possibly due to the increased melting performance and stickiness with time in the cheese [5]. 4 Conclusions Our findings result in four major conclusions. First, the LF without fat replacer was harder, less sticky, and had high stretch force and low meltability, as well as poor pizza bake and stretch performance. Second, these appearance defects were partially corrected via the addition of whey protein concentrates. Third, cheese cooking time was reduced from 2.5 h to approximately 50 min by reducing the pH of cheese milk from 5.9 to 5.5. Pre-acidification modified the functionality of Mozzarella cheeses, but excessive pre-acidification (pre-acidification of cheese milk to pH 5.5 or lower) negatively affected the stickiness, and pizza bake, and stretch performance of Mozzarella cheeses. Fourth, as aging progressed, the meltability, stickiness and stretch performance of Mozzarella cheese improved, whereas the hardness and stretch force of Mozzarella cheese decreased. The pizza bake test showed that Mozzarella cheese exhibited improved fluidity and less scorched shreds after a period of storage. These results indicated that many factors needed to be considered in the development of LF with whey protein as fat replacer in the dairy industry. Declarations Acknowledgments We are grateful to Arla Foods Amba (Copenhagen, Denmark) for funding this project via Grant 200008493.078148.26000.400.01. Availability of data The data that support the findings of this study are available on request from the corresponding author. Conflict of interest All authors have no conflict of interest. Author contributions Deju Zhang: Investigation; Methodology; Validation; Conceptualization; Formal analysis; Data curation; Writing-original draft. Juan Xiong: Formal analysis; Data curation; Validation; Writing-review & editing. Kai Jiang: Writing-review & editing. Xiaorui Zhao: Data curation; Formal analysis. Yiming Gan: Writing-review & editing. References 1. Akhtar, A.; Nasim, I.; Din, M.S.U.; Araki, T.; Khalid, N. Effects of different fat replacers on functional and rheological properties of low-fat mozzarella cheeses: A review. Trends in Food Science & Technology 2023 , 139 , doi:10.1016/j.tifs.2023.104136. 2. Matsuda, M.; Shimomura, I. Increased oxidative stress in obesity: Implications for metabolic syndrome, diabetes, hypertension, dyslipidemia, atherosclerosis, and cancer. Obesity Research & Clinical Practice 2013 , 7 , E330-E341, doi:10.1016/j.orcp.2013.05.004. 3. Zhang, D.J.; Lillevang, S.K.; Shah, N.P. 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Liu, J.; Han, L.; Cheng, D.; Li, S.; Chen, X.; Yu, Y.; Zhang, D.; Zhang, T.J.F.C. Incorporating ε-polylysine hydrochloride, tea polyphenols, nisin, and ascorbic acid into edible coating solutions: Effect on oxidation and structure of marinated egg proteins. Food Control 2024 , 110395. 18. Zhang, T.; Tang, Y.H.; Ge, H.F.; Zhang, D.J.; Li, T.; Cheng, D.K.; Liu, J.B.; Yu, Y.D. Storage impact on egg white powder's physical and functional properties. Journal of the Science of Food and Agriculture 2023 , 103 , 3799-3811, doi:10.1002/jsfa.12274. 19. Guinee, T.P.; Feeney, E.P.; Auty, M.A.E.; Fox, P.F. Effect of pH and calcium concentration on some textural and functional properties of Mozzarella cheese. Journal of Dairy Science 2002 , 85 , 1655-1669, doi:10.3168/jds.S0022-0302(02)74238-0. 20. Joshi, N.S.; Muthukumarappan, K.; Dave, R.I. Effect of calcium on microstructure and meltability of part skim Mozzarella cheese. 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Experimental evidence for previously unclassified calcium phosphate structures in the casein micelle. Journal of Dairy Science 2017 , 100 , 6938-6948, doi:10.3168/jds.2017-12623. 25. Dalgleish, D.G. On the structural models of bovine casein micelles-review and possible improvements. Soft Matter 2011 , 7 , 2265-2272, doi:10.1039/c0sm00806k. 26. Hennelly, P.J.; Dunne, P.G.; O'Sullivan, M.; O'Riordan, D. Increasing the moisture content of imitation cheese: effects on texture, rheology and microstructure. European Food Research and Technology 2005 , 220 , 415-420, doi:10.1007/s00217-004-1097-9. 27. Fife, R.L.; McMahon, D.J.; Oberg, C.J. Functionality of low fat Mozzarella cheese. Journal of Dairy Science 1996 , 79 , 1903-1910, doi:10.3168/jds.S0022-0302(96)76559-1. 28. Ismail, M.; Ammar, E.; El-Metwally, R. Improvement of low fat mozzarella cheese properties using denatured whey protein. International Journal of Dairy Technology 2011 , 64 , 207-217, doi:10.1111/j.1471-0307.2010.00654.x. 29. Yazici, F.; Akgun, A. Effect of some protein based fat replacers on physical, chemical, textural, and sensory properties of strained yoghurt. Journal of Food Engineering 2004 , 62 , 245-254, doi:10.1016/s0260-8774(03)00237-1. 30. Chevanan, N.; Muthukumarappan, K.; Upreti, P.; Metzger, L.E. Effect of calcium and phosphorus, residual lactose and salt-to-moisture ratio on textural properties of cheddar cheese during ripening. Journal of Texture Studies 2006 , 37 , 711-730, doi:10.1111/j.1745-4603.2006.00080.x. 31. Hinrichs, J. Incorporation of whey proteins in cheese. International Dairy Journal 2001 , 11 , 495-503, doi:10.1016/s0958-6946(01)00071-1. 32. Cunha, C.R.; Dias, A.I.; Viotto, W.H. Microstructure, texture, colour and sensory evaluation of a spreadable processed cheese analogue made with vegetable fat. Food Research International 2010 , 43 , 723-729, doi:10.1016/j.foodres.2009.11.009. 33. Fang, T.Q.; Shen, X.; Hou, J.C.; Guo, M.R. Effects of polymerized whey protein prepared directly from cheese whey as fat replacer on physiochemical, texture, microstructure and sensory properties of low-fat set yogurt. Lwt-Food Science and Technology 2019 , 115 , doi:10.1016/j.lwt.2019.108268. Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-4055582\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":278106688,\"identity\":\"adbb6b0b-f027-4ac4-bec6-3789de5f407d\",\"order_by\":0,\"name\":\"Deju Zhang\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIie3PIWsDMRTA8YSDU9nOvpJ9iBsHYWL0s2QEXk1FZcXYUpOZwez1W0xOpgRSE6g9UVMK85Uzxy4wZkrazk3kLwIJ/Mh7hORy/7Fqod1X3z+//b5ASQjVMk3ArfaktHSpf+7nCUHVRPJuLyW1DYLP2LZo1s7vZ/PxE+EGgX5gmrhXwVv4LEXASdMGBeTGe6BhevIXYHXBRMcEvzJ2GGxigJr5CTIdiCygaSPpLyLD+sy6uoZIdCQ4DGbSg406t9otNUoIiJx5NTKA6u7BpNe/3iy0Peh7Wb04z9njuKoAb7uDUUlyXBkP+QeQy+VyueO+ARaoUvnMvL/nAAAAAElFTkSuQmCC\",\"orcid\":\"\",\"institution\":\"The University of Hong Kong\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Deju\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":278106689,\"identity\":\"0694bc4c-8168-423e-b55b-51e11b791f6c\",\"order_by\":1,\"name\":\"Juan Xiong\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Hengyang Medical School\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Juan\",\"middleName\":\"\",\"lastName\":\"Xiong\",\"suffix\":\"\"},{\"id\":278106690,\"identity\":\"0d807c9f-2613-477d-8350-0c6659f42de4\",\"order_by\":2,\"name\":\"Kai Jiang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Northeastern University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kai\",\"middleName\":\"\",\"lastName\":\"Jiang\",\"suffix\":\"\"},{\"id\":278106691,\"identity\":\"2dfa580f-2869-4748-92a3-697dce37efac\",\"order_by\":3,\"name\":\"Xiaorui Zhao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Aarhus University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiaorui\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"},{\"id\":278106692,\"identity\":\"bddb17f5-703b-4fad-8e3a-f3c90300ba96\",\"order_by\":4,\"name\":\"Yiming Gan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"The University of Hong Kong\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yiming\",\"middleName\":\"\",\"lastName\":\"Gan\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-03-09 11:50:12\",\"currentVersionCode\":2,\"declarations\":{\"humanSubjects\":false,\"vertebrateSubjects\":false,\"conflictsOfInterestStatement\":false,\"humanSubjectEthicalGuidelines\":false,\"humanSubjectConsent\":false,\"humanSubjectClinicalTrial\":false,\"humanSubjectCaseReport\":false,\"vertebrateSubjectEthicalGuidelines\":false},\"doi\":\"10.21203/rs.3.rs-4055582/v2\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4055582/v2\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":52630412,\"identity\":\"1c06a81b-671e-41b8-8382-e872dcb17556\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":169024,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eColor and browning of Mozzarella cheeses made from milk pre-acidified to different pH\\u003c/p\\u003e\\n\\u003cp\\u003e-c and -b represent the color and browning values of Mozzarella cheese made from milk pre-acidified to different pH, respectively. A is the color and browning values of FF9, LF9, LFC9, and LFY9; B is the color and browning values of FF8, LF8, LFC8, and LFY8; C is the color and browning values of FF7, LF7, LFC7, and LFY7; D is the color and browning values of LF6, LFC6, and LFY6; E is the color and browning values of LF5, LFC5, and LFY5.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/d4030a1d7582e23d43ab3742.png\"},{\"id\":52630410,\"identity\":\"ebc32b41-2f08-49e2-9020-1aa759fb3254\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:43\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":262570,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCalcium content of Mozzarella cheeses made from milk pre-acidified to different pH\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/943921af992bffbb624eb825.png\"},{\"id\":52630417,\"identity\":\"e008ef34-6cc8-4664-b9d4-5f62bec50181\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":188084,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMelt length and meltability of low-fat Mozzarella cheeses made from milk pre-acidified to different pH\\u003c/p\\u003e\\n\\u003cp\\u003e-l and -a represent the melt length and meltability values of Mozzarella cheese made from milk pre-acidified to different pH, respectively. A is the melt length and meltability values of FF9, LF9, LFC9, and LFY9; B is the melt length and meltability values of FF8, LF8, LFC8, and LFY8; C is the melt length and meltability values of FF7, LF7, LFC7, and LFY7; D is the melt length and meltability values of LF6, LFC6, and LFY6; E is the melt length and meltability values of LF5, LFC5, and LFY5.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/040f71e68d01a41903b187c6.png\"},{\"id\":52630418,\"identity\":\"73d84511-9b05-4f8d-824f-ba09ced30764\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1051843,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePizza bake performance of full-fat and low-fat Mozzarella cheeses made from milk pre-acidified to pH 5.8 and pH 5.9\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/18ee54db8d1338acf9a01d9a.png\"},{\"id\":52630419,\"identity\":\"1e3bfd84-e148-4e7e-891f-b03812d289bf\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":732579,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003ePizza bake performance of full-fat and low-fat Mozzarella cheeses made from milk pre-acidified to pH 5.5, pH 5.6, and pH 5.7\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/3c04b0131c3936b13dd7ac34.png\"},{\"id\":52630415,\"identity\":\"9b8e0c40-3d50-4dc0-b13e-daca992baad6\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":81098,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eHardness of Mozzarella cheeses made from milk pre-acidified to different pH\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA is the hardness values of FF9, LF9, LFC9, and LFY9; B is the hardness values of FF8, LF8, LFC8, and LFY8; C is the hardness values of FF7, LF7, LFC7, and LFY7; D is the hardness values of LF6, LFC6, and LFY6; E is the hardness values of LF5, LFC5, and LFY5.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/bc204fb0aa5d742326158b1b.png\"},{\"id\":52630416,\"identity\":\"f5bf1740-7408-4434-a462-7edfe741a5c4\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":76757,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStickiness of Mozzarella cheeses made from milk pre-acidified to different pH\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA is the stickiness values of FF9, LF9, LFC9, and LFY9; B is the stickinessvalues of FF8, LF8, LFC8, and LFY8; C is the stickiness values of FF7, LF7, LFC7, and LFY7; D is the stickinessvalues of LF6, LFC6, and LFY6; E is the stickinessvalues of LF5, LFC5, and LFY5.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/6ddb722c83c741ffe529aaca.png\"},{\"id\":52630414,\"identity\":\"882fe9b5-ea18-40ee-bc0e-97e214fd3a28\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":74084,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStretch force of Mozzarella cheeses made from milk pre-acidified to different pH\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA is the force values of FF9, LF9, LFC9, and LFY9; B is the force values of FF8, LF8, LFC8, and LFY8; C is the forcevalues of FF7, LF7, LFC7, and LFY7; D is the force values of LF6, LFC6, and LFY6; E is the force values of LF5, LFC5, and LFY5.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/0ad36f5d234e0e2c2109e73f.png\"},{\"id\":52630413,\"identity\":\"5c77a3c5-86db-4ac2-b669-c62e0bf16cb0\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:45\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":882473,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStretch performance of low-fat Mozzarella cheeses made from milk pre-acidified to pH 5.8 and pH 5.9\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/e26ae295a0d48b7a20b80175.png\"},{\"id\":52630420,\"identity\":\"580e466a-d10e-4ce2-ae27-edb08d9dec13\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:23:46\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":693589,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eStretch performance of low-fat Mozzarella cheeses made from milk pre-acidified to pH 5.5, pH 5.6, and pH 5.7\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/5c488a5a945fa120debf9571.png\"},{\"id\":52630946,\"identity\":\"13a71499-7895-4d11-9ae6-4e8a4cfcdb85\",\"added_by\":\"auto\",\"created_at\":\"2024-03-13 19:31:45\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":4455554,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4055582/v2/618666e4-13c7-4f12-9df5-621a3477b5a2.pdf\"}],\"financialInterests\":\"The authors declare no competing interests.\",\"formattedTitle\":\"Effect of pre-acidification and whey proteins on low-fat Mozzarella cheese during storage: physicochemical, textural, and functional properties\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eFats have many functional properties in a food carrier as they play a significant role in the texture, color, and sensory properties of foods in many specific ways\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[1]. However, excessive consumption of fat is related to a lot of health risks, such as obesity, atherosclerosis, hypertension, diabetes, inflammation, gall bladder disease, and cancer\\u0026nbsp;[2]; this has aroused the concern of consumers that dietary fat intake should be tightly controlled to reduce the risk of multiple diseases\\u0026nbsp;[3]. Driven by the expansion of the pizza industry, Mozzarella cheese sales achieve substantial growth and currently become the most commonly purchased cheese in the US\\u0026nbsp;[4]. Full-fat Mozzarella cheese (FF) is conventionally used as a topping on pizza, because of its good melting and stretching properties\\u0026nbsp;[3]. But they may contain more than 20% fat, which indicates a health threat to cheese lovers\\u0026nbsp;[5].\\u003c/p\\u003e\\n\\u003cp\\u003eIn view of the current situation, many studies were conducted to study the functionality of low-fat Mozzarella cheese (LF). The reduction of fat in LF could cause a reduction in oiling off and moisture to protein values, which gives the cheeses poor functionality and limits their use on pizza\\u0026nbsp;[6]. Moreover, fat-reduced cheese is conventionally low in whiteness values and may exhibit an unappealing appearance\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[5]. To overcome these drawbacks, several studies were carried out to improve the quality of LF, such as the use of pre-acidification, EPS-producing culture, and fat replacer\\u0026nbsp;[3,7]. Among them, whey protein concentrates are commonly used due to their high nutritional value and good functionality, such as foaming, gelling and emulsifying properties\\u0026nbsp;[8], which allows them to be used as fat replacers in many food systems\\u0026nbsp;[9]. Low-fat dairy products with whey protein may exhibit comparable functional properties to their full-fat counterparts. Therefore, determining the functionality of low-fat cheeses containing whey protein concentrates (WPC) is beneficial for promoting the quality of LF and providing a reference for the utilization of whey protein in the cheese industry.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePre-acidification also plays an important role in Mozzarella cheese functionality and texture by affecting the cross-linking between calcium and protein\\u0026nbsp;[10]. High levels of bound calcium in LF are positively related to denser cheese structure and higher values of hardness\\u0026nbsp;[6]. Pre-acidification treatment could remove bound calcium from casein micelles and reduce the cross-linking of casein, substantially producing a low-fat cheese with improved qualities\\u0026nbsp;[10]. However, over-acidification results in excessive bound calcium being released from the curd, thus yielding cheese with excessive softness and high flowability. It is also not acceptable for Mozzarella cheese to be used as a pizza topping.\\u003c/p\\u003e\\n\\u003cp\\u003eThus, the aim of this study was to investigate the effect of combining the technique of pre-acidification and the use of whey protein concentrates on hardness, melt, color, stretch properties and pizza bake properties of Mozzarella cheese. As a result, the best final pH of pre-acidification was selected for follow-up studies. Specifically, FF, LF, and LF supplemented with whey protein concentrate (LFWP) were determined for evaluating the potential of LFWP to be used in pizza industry.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003ch3\\u003e\\u003cstrong\\u003e2.1\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eMaterials\\u0026nbsp;\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003ePasteurized and homogenized milk (full-fat milk: fat content: 5.1 g/100 mL, low-fat milk: fat content: 2.5 g/100 mL, and skimmed milk: fat content: 0.7 g/100 mL) were purchased from local distributors (Farm Milk Company Limited, Hong Kong). Both the Nutrilac\\u0026reg; CH-4560 and Nutrilac\\u0026reg; YO-8075 were commercial whey protein concentrates and were supplied by Arla Food Amba (Copenhagen, Denmark). Double-strength chymosin was purchased from Chr. Hansen, Bayswater, Victoria, Australia. \\u003cem\\u003eStreptococcus thermophilus\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e1275 (ST)\\u003cem\\u003e, Lactobacillus delbrueckii\\u0026nbsp;\\u003c/em\\u003essp\\u003cem\\u003e. bulgaricus\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e859 (LB), \\u003cem\\u003eLactobacillus casei\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e290 (LC), and \\u003cem\\u003eLactobacillus paracasei\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e276 (LP) were obtained from the Australian Starter Culture Research Center (ASCRC, Werribee, Australia).\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.2 Culture propagation\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eAll microorganisms were stored in sterile MRS broth with 50 g/L glycerol at -80 ℃. To activate the ST, LB, LC, and LP cultures, a 100 \\u0026mu;L aliquot of each culture was individually transferred into MRS broth and incubated in a CO\\u003csub\\u003e2\\u003c/sub\\u003e incubator (Shel-Lab Co., Cornelius, U.S.A.) at 37 ℃ for 18 h following the method of\\u0026nbsp;Zhang\\u003cem\\u003e, et al.\\u003c/em\\u003e [11]. After the second transfer in MRS broth, the activated starter cultures were transferred into 10% (w/w) reconstituted skim milk for another two transfers. Activated cultures were used for cheese making.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.3 Cheese making\\u003c/strong\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eMozzarella cheeses were made following the method of Ayyash and Shah [12] with some modifications. An aliquot of 2 L fresh milk was cultured with 1 mL/100 mL inoculum of each bacterial strain and acidified with 100 mM citric acid to pH 5.9, 5.8, 5.7, 5.6, and 5.5 at 37 ℃ in a custom-made, temperature-controlled cheese vats. Eighteen batches of Mozzarella cheeses were manufactured as indicated below.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp style='margin-right:0in;margin-left:0in;text-align:justify;font-size:14px;font-family:\\\"Times New Roman\\\",serif;font-weight:bold;line-height:200%;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;font-weight:bold;'\\u003eTable 1 Mozzarella cheeses made from milk pre-acidified to different pH\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003ctable style=\\\"width: 4.2e+2pt;margin-left:12.15pt;border-collapse:collapse;border:none;\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.65pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eNo.\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.65pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eCheese\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.65pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eFat content (g/100 g milk)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.65pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003ePreacidification pH\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:24.65pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eFat replacer\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:14.95pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e1\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:14.95pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eFF9\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:14.95pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e2.21\\u003c/span\\u003e\\u003c/p\\u003e\\n 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style='font-family:\\\"Times New Roman\\\",serif;'\\u003e5.5\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:17.1pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eCH-4560\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:17.1pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e18\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:17.1pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eLFY5\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:17.1pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e1.10\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:17.1pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e5.5\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:84.7pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:17.1pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eYO-8075\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;text-indent:21.0pt;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eThe curd made from high-fat milk (fat content: 2.21 g/100 g) pre-acidified to pH 5.6 or 5.5 was too thin and can not be piled into slabs. \\u003cem\\u003eStreptococcus thermophilus\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e1275\\u003cem\\u003e, Lactobacillus delbrueckii\\u0026nbsp;\\u003c/em\\u003essp\\u003cem\\u003e. bulgaricus\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e859, \\u003cem\\u003eLactobacillus casei\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e290, and \\u003cem\\u003eLactobacillus paracasei\\u0026nbsp;\\u003c/em\\u003eASCC\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e276 were used in all treatments.\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNutrilac\\u0026reg; CH-4560 and Nutrilac\\u0026reg; YO-8075 (10 g) were added directly to cheese milk and mixed for 1 min. After 40 min of aging, 1 mL chymosin diluted in 19 mL distilled water was added to the cheese milk, followed by a brief stirring. The milk got coagulated after 45 min and then it was cut into 1 cm\\u003csup\\u003e3\\u003c/sup\\u003e cube with a knife. The cut curd was cooked as described by\\u0026nbsp;Dai\\u003cem\\u003e, et al.\\u003c/em\\u003e [13]\\u0026nbsp;at 37 ℃ until the pH of the curd dropped to 5.3. During this time, whey was drained off and the cooked curd was piled and chopped into slabs. Thereafter, a salting process (1.5 g/100 g) followed and curds were mellowed for another 20 min. All cheeses were hand-stretched in hot brine solution (4 g/100 g) maintained at 75 ℃ and then kneaded in blocks. After stretching and kneading, the 200 g blocks of Mozzarella cheese were vacuum packaged and aged at 4 ℃ for 2 weeks. Sampling was carried out to determination of functionality and chemical composition on days 0, 7, and 14 of storage.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.4 Composition\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eMoisture was determined by a moisture analyzer (HB43-S, Mettler Toledo, Switzerland), protein by Kjeldahl method, and fat by Babcock method as described by\\u0026nbsp;Dai, Jiang, Corke and Shah [13]\\u0026nbsp;and\\u0026nbsp;Liu\\u003cem\\u003e, et al.\\u003c/em\\u003e [14]. The cheese yield was calculated as the weight of total cheese to the weight of total materials, multiplied by 100. All analyses were performed in triplicate.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.5 Color\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe color parameters of cheeses were analyzed with a Minolta Chroma Meter (CR-300 Series, Minolta, Japan) following the method of\\u0026nbsp;Zhao\\u003cem\\u003e, et al.\\u003c/em\\u003e [15]. The surface color of cheeses was taken at 4 external and 4 internal locations of the cheeses on days 0, 7, and 14 of storage. An aliquot of the ground cheese (15 g) was weighed into a disk (7.5 cm \\u0026times; 7.5 cm \\u0026times; 1.5 cm) and was heated at 100 ℃ for 160 min. After cooling, the color of cheeses was measured again, this represented the browning of cheeses. The color and browning of cheese were expressed as L* (0-100, black to white), a* (green (-) to red (+)), and b* ( blue (-) to yellow (+)).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.6 Calcium\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eCheese (0.4 g) with 2 mL of 30 % w/w H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e and 6 mL of nitric acid was digested using a Microwave Digester (ETHOS-1600, Milestone, Italy) for 90 min. Afterward, the calcium content of cheese was analyzed using an atomic absorption spectrophotometer (iCE 3300, Thermo Scientific, UK) with an air (13.5 L/min)/acetylene (2.0 L/min) flame as indicated by\\u0026nbsp;Zisu and Shah [10]. The calcium atomic spectroscopy standard containing 993 \\u0026mu;g/mL calcium (Sigma‐Aldrich, St. Louis, MO, USA) was applied to prepare working standards in a sample buffer (49.2% deionized water, 48.7% of 12% (w/v) trichloro-acetic acid and 2.1% of 5% (w/v) lanthanum oxide).\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.7 Meltability\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe meltability of cheeses was determined according to the method of\\u0026nbsp;Cais-Sokolinska\\u003cem\\u003e, et al.\\u003c/em\\u003e [16]\\u0026nbsp;with some modifications. Grated cheese (10 g) was placed in a glass tube (30 mm \\u0026times; 200 mm) and packed to form a plug at the bottom. The cheeses were heated at 100 ℃ for 12 min and the length of the cheese was measured before heating and after the cheeses were cooled down to room temperature. The meltability of cheese was expressed as the length of cheese after heating minus the length of cheese before heating and the percentage meltability was calculated as length difference divided by the initial length of cheese and multiplied by 100%.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.8 Pizza bake\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003ePizza dough was manually made and baked as described by\\u0026nbsp;Zhang, Ling, Jiang, Zhao and Gan [7]. Then cheese (20 g) was cut into shreds (approximetely 2 mm \\u0026times; 2 mm \\u0026times; 20 mm pieces) using a cheese grater and spread on the top of the pizza base, followed by baking at 220 ℃ for 5 min. The changes in Mozzarella cheese were observed visually for shred fusion, melting and scorching degree, and extent of blistering.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.9 Texture\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe hardness and stickiness of Mozzarella cheeses were determined using a TA.XT Plus model texture analyzer (Stable Micro Systems, Godalming, Surrey, UK) following the method of\\u0026nbsp;Liu\\u003cem\\u003e, et al.\\u003c/em\\u003e [17]\\u0026nbsp;and\\u0026nbsp;Zhang\\u003cem\\u003e, et al.\\u003c/em\\u003e [18]\\u0026nbsp;with modifications. The Mozzarella cheeses were cut into cubes (30 mm \\u0026times; 30 mm \\u0026times; 20 mm) and placed on the stage of the texture analyzer. The TPA conditions were set as follows: test speed of 3.0 mm/s, and test distance of 20 mm.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe stretch force and stretch performance of the melted cheese were also determined following the method of\\u0026nbsp;Dai, Jiang, Corke and Shah [13]. Cheese samples were cut into small cubes and filled evenly in a plastic pot with a fork. Then the sample pot with fork and cheese cubes was heated in a microwave oven (600 W) for 40 s and tested immediately after the temperature of the melted cheese reached 55 ℃. The TPA conditions were set as follows: test speed of 20 mm/s and test distance of 270 mm. The stretch performance of Mozzarella cheeses was recorded using a camera.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e2.10 Statistical data evaluation\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eFor each assay, at least three independent samples were prepared. Results were given as mean \\u0026plusmn; standard deviation (SD) (n = 3). Figures in this chapter were drawn with GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).\\u003c/p\\u003e\"},{\"header\":\"3 Results and discussion\",\"content\":\"\\u003ch3\\u003e\\u003cstrong\\u003e3.1 Composition\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eTable 2 shows the composition of FF and LF. The protein and moisture content of FF were at low level (protein content was 16.44%, 16.06%, and 15.37%, and moisture content at D 0 was 58.07%, 59.93%, and 60.93% for FF9, FF8, and FF7, respectively) compared to LF and LFWP, and it was expected due to the hydrophobicity of fat globules. As the pre-acidified pH of cheese milk decreased, the yield and moisture content of Mozzarella cheese increased, while the fat and protein content decreased. This is perhaps because the removal of bound calcium from casein micelles caused by pre-acidification increased the hydration of the cheese para-casein matrix, thereby increasing the moisture content of cheese [19,20]. The moisture and yield of LFWP were higher than that of the LF, corresponding to the fact that whey protein has excellent moisture retention capacity. Finally, we also found that the moisture of cheese showed fluctuation in the range of 58.22% to 65.23% and remained stable over storage time.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003cp style='margin-right:0in;margin-left:0in;text-align:justify;font-size:14px;font-family:\\\"Times New Roman\\\",serif;font-weight:bold;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;font-weight:bold;'\\u003eTable 2 Yield and composition of Mozzarella cheese\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003ctable style=\\\"width:465.35pt;margin-left:12.15pt;border-collapse:collapse;border: none;\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 41.8pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eCheese\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 66.6pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eYield (%)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 69.3pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eFat (%)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 68pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eProtein (%)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 66.3pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eMoisture (%, D 0)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 77.35pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eMoisture (%, D 7)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 76pt;border-width: 1pt medium;border-style: solid none;border-color: windowtext currentcolor;padding: 0in 5.4pt;height: 15.6pt;vertical-align: top;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eMoisture (%, D 14)\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width:41.8pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eFF\\u003c/span\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e9\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:66.6pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e13.17 \\u0026plusmn; 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0.07\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:69.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e7.25 \\u0026plusmn; 0.64\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:68.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e18.76 \\u0026plusmn; 0.90\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:66.3pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;vertical-align:middle;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e65.5 \\u0026plusmn; 1.63\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:77.35pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;vertical-align:middle;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e65.69 \\u0026plusmn; 0.87\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:76.0pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;vertical-align:middle;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e65.23 \\u0026plusmn; 0.47\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width:41.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eLFY5\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:66.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e12.73 \\u0026plusmn; 0.37\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:69.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e7.20 \\u0026plusmn; 0.60\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:68.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e18.78 \\u0026plusmn; 0.98\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:66.3pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;vertical-align:middle;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e65.34 \\u0026plusmn; 1.38\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:77.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;vertical-align:middle;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e65.40 \\u0026plusmn; 1.07\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width:76.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;height:15.6pt;\\\"\\u003e\\n \\u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;vertical-align:middle;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003e65.01 \\u0026plusmn; 0.44\\u003c/span\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\\\"Calibri\\\",sans-serif;text-indent:21.0pt;'\\u003e\\u003cspan style='font-family:\\\"Times New Roman\\\",serif;'\\u003eValues are expressed as mean \\u0026plusmn; standard deviation.\\u003c/span\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e3.2 Color and browning\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe color and browning values of Mozzarella cheeses during\\u0026nbsp;storage\\u0026nbsp;are given in Figure 4.1.\\u0026nbsp;The browning L* values of Mozzarella cheese were lower compared to the color L* values of Mozzarella cheeses. Besides, it was found that the color and browning values of FF were higher than those of LF and LFWP during the entire period of storage. This difference can be interpreted as fat globules contribute to the lightness of cheeses by scattering light\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[21]. As expected, significant differences were found in color and browning values between LF and LFWP, showing that the addition of whey protein in Mozzarella cheeses improved the whiteness of Mozzarella cheese before and after baking. The improved color L* values in LFWP might be attributed to more light penetrating the superficial layers is scattered by the edges of the whey pockets [22]. The higher browning L* values of LFWP were possibly due to free water in cheese that can protect the casein matrix from dehydration during baking by adsorbing heat energy. In addition, results also showed that a decrease in the final pH of pre-acidified cheese milk induced an increase in the color and browning values of cheeses, so in the LFC5 and LFY5, these two parameters were close to those of the FF9. We also observed a reduction of color L* values and an increase of browning L* values as cheese age increased. The former was because of serum phase and proteolytic changes during storage [13], and the latter was because the active metabolism of starter bacteria reduced the reducing sugar content, further decreasing the formation of Maillard reactions products that might affect the whiteness of Mozzarella cheese.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e3.3 Calcium\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eFigure 4.2 revealed progressively declining values of the calcium content of Mozzarella cheese in response to increasing amount of citric acid added to cheese milk. Solubilization of casein micelles resulting from acidification leads to dissociation of colloidal calcium phosphate, and this solubilization will lead to the release of calcium in whey, which explains why a decreased trend in calcium content was observed as the final pH of pre-acidification of cheese milk decreased\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[23].\\u003c/p\\u003e\\n\\u003cp\\u003eThe calcium content of LF and LFWP was higher compared to FF. As suggested by\\u0026nbsp;Hindmarsh and Watkinson [24]\\u0026nbsp;and\\u0026nbsp;Dalgleish [25], the subunits of casein micelle are bound together through calcium phosphate bridges via van der waals forces, hydrophobic bonds, and ionic calcium linkages, which contribute to enhanced calcium content in casein matrix. Therefore, the higher calcium content in LF and LFWP might result from their higher protein content. No significant difference was observed in calcium content between LF and LFWP. Our results were in agreement with the conclusions of\\u0026nbsp;Zisu and Shah [10], who also saw a decrease in calcium content when cheese milk was pre-acidified with citric acid. However, there is a great difference in calcium content compared to the current investigation. This may be because of the difference in protein content between their cheeses and ours.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e3.4 Meltability\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe observed changes in melt length and meltability during storage are shown in Figure 4.3. The average melt length of FF9, FF8, and FF7 at D 0 was 4.25 cm, 6.88 cm, and 12.30 cm compared to 0.78 cm, 2.16 cm, 5.22 cm, 7.32 cm, and 11.47 cm for LF9, LF8, LF7, LF6, and LF5, respectively. Similar results were also observed for the meltability values of Mozzarella cheeses. As the proportion of protein increases, the calcium phosphate para-casein network becomes more fused and contiguous, resulting in the displacement of adjoining planes. Thereafter, the mobility of the cheese matrix became more difficult especially as the level of free oil decreases simultaneously [5]. In addition, the higher calcium content in LF also has a negative impact on their melt length and meltability [19]. Due to these main reasons, the melt length and meltability of Mozzarella cheeses were greatly reduced in LF and LFWP.\\u003c/p\\u003e\\n\\u003cp\\u003eIt was observed that the melt length and meltability of Mozzarella cheese enhanced as the final pH of pre-acidification decreased. There are two main reasons for this. Pre-acidification increased the moisture content of the protein matrix, which further attenuates protein-protein interactions thus plasticizing the matrix, resulting in higher mobility\\u0026nbsp;[26]. Secondly, the removal of calcium resulting from pre-acidification causes increased interaction of proteins with surrounding serum, resulting in higher levels of protein hydration\\u0026nbsp;[20]. A direct relationship between calcium content, meltability and texture of cheese has also previously been described by\\u0026nbsp;Joshi, Muthukumarappan and Dave [20]\\u0026nbsp;and\\u0026nbsp;Fife\\u003cem\\u003e, et al.\\u003c/em\\u003e [27]. Cheeses containing high calcium content were surrounded by more free serums, while cheeses with low calcium content have less as these free serums are partially absorbed by the protein matrix. When free serums were absorbed by proteins, the proteins become more hydrated, which gives the protein matrix higher flowability during heating.\\u003c/p\\u003e\\n\\u003cp\\u003eThe influence of WPC on the meltability of cheeses was also studied in this exploration. The addition of whey protein to cheese milk increased the melt length and meltability of Mozzarella cheese, and the effect of whey proteins of CH-4560 and YO-8075 treatments on Mozzarella cheese was similar. In the study of\\u0026nbsp;Ismail\\u003cem\\u003e, et al.\\u003c/em\\u003e [28], they attributed this to high moisture retention of Mozzarella cheeses caused by WPCs. However, we also hold that WPCs serve as fat replacers and lubricate the casein matrix. And it could also mimic the emulsified fat via forming a dispersed phase of particles that are free to move independently\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[29]. These action ways allow Mozzarella cheeses containing whey protein have better melting performance.\\u003c/p\\u003e\\n\\u003cp\\u003eThe melt length and meltability of Mozzarella cheeses increased during cold storage, and these two parameters exceeded the limit of the test (the maximum melting length is 14.5 cm) in LFC6, LFY6, and LF5 at D 7 and D 14, and in LFC5 and LFY5 at D 0, D 7, and D 14. The above trends also concur with those of others\\u0026nbsp;[5,27], who also found that a period of\\u0026nbsp;storage\\u0026nbsp;increased the melt length and meltability of Mozzarella cheese. The greater melting performance might be due to degradation of the casein matrix\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[5].\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e3.5 Pizza bake\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eThe pizza bake images of Mozzarella cheeses are shown in Figure 4.4 and Figure 4.5. After baking, FF9, LF9, LFC9, LFY9, LF8, LFC8, LFY8, LF7, LFC7, and LFY7 showed incomplete shred fusion with low mobility at D 0, which is because of the relatively high final pH of pre-acidification and the lower fat content in cheeses. However, FF8, FF7, LF6, LFC6, LFY6, LF5, LFC5, and LFY5 exhibited a much better ductility and meltability, showing that pre-acidification played a significant role in the pizza bake performance of Mozzarella cheeses. It was found that LF9 exhibited scorch formation and created an unappealing surface at D 14, but the scorching decreased with the introduction of WPCs.\\u003c/p\\u003e\\n\\u003cp\\u003eBy comparison, we can see that the LFC8 and LFY8 showed better pizza bake performance with improved melting and ductility compared to that of the FF8 and LF8 at D 0 and D 14. Similar conclusions can also be drawn by comparing the LF7 with LFC7 and LFY7, and LF6 with LFC6 and LFY6. Additionally, when the pre-acidification pH of cheese milk was no greater than 5.7, Mozzarella cheese with added whey protein demonstrated comparable baking properties to full-fat cheeses. All the Mozzarella cheeses made from cheese milk pre-acidified to pH 5.5 exhibited high meltability and flowability. However, these Mozzarella cheeses had an excessively thin melting texture, which may cause poor taste and greatly affect consumer preference.\\u003c/p\\u003e\\n\\u003cp\\u003eAt D 14, all the Mozzarella cheese showed more complete shred fusion than at D 0. This observation was similar with the changing pattern of meltability of Mozzarella cheeses.\\u0026nbsp;Dai, Jiang, Shah and Corke [5]\\u0026nbsp;also observed incompletely shred fusion and some scorched shreds around the edge of the pizza in LFC at D 7, and these cheeses exhibited improved melting performance and less scorching after 28 days of storage. The author also reported that low-fat Mozzarella cheese showed big blister after baking at D 28, but in this study, we did not observe this phenomenon in these Mozzarella cheeses. This may be because our cheese sticks before baking were very thin, which avoided the formation of blisters in the cheese.\\u003c/p\\u003e\\n\\u003ch3\\u003e\\u003cstrong\\u003e3.6 Texture\\u003c/strong\\u003e\\u003c/h3\\u003e\\n\\u003cp\\u003eTexture values are an important indicator of the textural characteristics of a cheese that correlates well with sensory scores\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[30]. Mozzarella cheese made with high-fat milk was softer than cheese made with low-fat milk (Figure 4.6). This is because the greater fat content in cheese, the more interruptions there are in the casein matrix and increased interference in the long-range interactions between the proteins. Therefore, the reduction of fat in cheese matrix allows protein interactions to be very extensive, leading to a rubbery texture\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[3].\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe addition of whey protein concentrates contributes to the reduction of hardness of Mozzarella cheeses. As discussed previously, the whey protein concentrates could retain a certain amount of water and be inserted inertly into the pores of the protein matrix like fat globules, which may disrupt the homogeneity of the protein network and further decrease the hardness of cheese\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[31]. Moreover, our results showed the hardness of cheeses decreased with the increasing amount of citric acid added to cheese milk. Specifically, at D 0, the hardness values of LF9, LFC9, and LFY9 were 778.85 g, 594.14 g, and 614.01 g, respectively, while these cheeses were associated with lower hardness values when made using cheese milk pre-acidified to pH 5.5 and their hardness values decreased to 141.50 g, 108.47 g, and 111.76 g for LF5, LFC5, and LFY5. After 14 days of storage, a slight decrease in the hardness of all the Mozzarella cheeses was observed, except for FF.\\u003c/p\\u003e\\n\\u003cp\\u003eThere was a significant difference between the Mozzarella cheeses for the parameter of stickiness (P \\u0026lt; 0.05). As indicated in Figure 4.7, FF possessed the highest stickiness among cheeses (absolute value, similarly hereinafter), while LF had the least hardness. The interactions between fat globules and casein increase the degree of adherence between cheeses and spherical probe with which it is in contact\\u003csup\\u003e\\u0026nbsp;\\u003c/sup\\u003e[32]. Furthermore, the incorporation of whey protein concentrates in cheese milk increased the stickiness of Mozzarella cheese. In our study, whey proteins of CH-4560 and YO-8075 were micro-particulated whey proteins. As suggested by\\u0026nbsp;Fang\\u003cem\\u003e, et al.\\u003c/em\\u003e [33], denatured whey protein concentrates could form a high molecular weight whey protein polymer similar to hydrocolloids, thus creating high level of cross-linking within the gel network, ultimately increasing the stickiness of dairy products.\\u003c/p\\u003e\\n\\u003cp\\u003eHigher stickiness values were noted as the final pre-acidification pH of Mozzarella cheeses decreased. This can be explained by the protein structure of the casein network. In terms of FF9, LF9, LFC9, and LFY9, they have highly aggregated protein network and there is more moisture present in serum channels. Therefore, the proteins tend to maintain their self-association and exhibited low adhesiveness. However, when a significant amount of calcium was removed from Mozzarella cheese due to pre-acidification of cheese milk to pH 5.5, the proteins are more unfolded, which allows them to interact with other surfaces. In our study, LF5, LFC5, and LFY5 had highly hydrated protein matrices and the cheese was very sticky before and after heating. Therefore, we can speculate that the unfolding of the protein aggregates in the low calcium conditions exposes more hydrophobic sites and charged sites, which gives Mozzarella cheeses a higher level of flexibility and allows the proteins to readily interact with the surface with which it is in contact. Furthermore, the stickiness of LF and LFWP declined with increasing storage time, but this downward trend did not constitute statistical significance (P \\u0026gt; 0.05).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe stretch force of Mozzarella cheeses with different final pre-acidification pH is shown in Figure 4.8. As the fat content of cheese decreased, the stretch force increased. The addition of whey protein concentrates resulted in a lower stretch force, this is especially evident in the Mozzarella cheeses made from milk pre-acidified to pH 5.9, 5.6, and 5.5. The results also showed that treatment with citric acid decreased the stretch force of Mozzarella cheeses, and the stretch force of melted cheese gradually decreased with the increasing amount of citric acid. Finally, aging also had a positive effect on the stretch force values of LF and LFWP, whereas FF9, FF8, and FF7 were not significantly affected by cold storage (P \\u0026gt; 0.05).\\u003c/p\\u003e\\n\\u003cp\\u003eThe stretch performance of FF, LF, and LFWP was also recorded and presented in Figure 4.9 and Figure 4.10. It is obvious that the Mozzarella cheeses made from milk pre-acidified to pH 5.6 and 5.5 had high moisture retention and exhibited smooth texture. These findings were consistent with the moisture content and the stretch force results. In terms of LF9, LFC9, LFY9, LF8, LFC8, LFY8, and LF7, the strands of these cheeses showed a rough texture. Combining the results here with the stretch force values, it can be concluded that the protein matrix of cheeses made from milk pre-acidified to high pH was tightly associated with each other, the bonds between proteins are stronger and require more energy to break.\\u003c/p\\u003e\\n\\u003cp\\u003eFrom these images, the Mozzarella cheese with WPCs had improved stretch performance with longer and less fractured strands were observed in LFWP than in LF, which may be attributed to the excellent thickening properties of whey proteins. With the increasing storage time, the Mozzarella cheeses exhibited more appealing stretch performance at d 28 as they showed longer and thicker strand formation compared to Mozzarella cheeses at d 0. This phenomenon was possibly due to the increased melting performance and stickiness with time in the cheese [5].\\u003c/p\\u003e\"},{\"header\":\"4 Conclusions\",\"content\":\"\\u003cp\\u003eOur findings result in four major conclusions. First, the LF without fat replacer was harder, less sticky, and had high stretch force and low meltability, as well as poor pizza bake and stretch performance. Second, these appearance defects were partially corrected via the addition of whey protein concentrates. Third, cheese cooking time was reduced from 2.5 h to approximately 50 min by reducing the pH of cheese milk from 5.9 to 5.5. Pre-acidification modified the functionality of Mozzarella cheeses, but excessive pre-acidification (pre-acidification of cheese milk to pH 5.5 or lower) negatively affected the stickiness, and pizza bake, and stretch performance of Mozzarella cheeses. Fourth, as aging progressed, the meltability, stickiness and stretch performance of Mozzarella cheese improved, whereas the hardness and stretch force of Mozzarella cheese decreased. The pizza bake test showed that Mozzarella cheese exhibited improved fluidity and less scorched shreds after a period of storage. These results indicated that many factors needed to be considered in the development of LF with whey protein as fat replacer in the dairy industry.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe are grateful to Arla Foods Amba (Copenhagen, Denmark) for funding this project via Grant 200008493.078148.26000.400.01.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe data that support the findings of this study are available on request from the corresponding author.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll authors have no conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eDeju Zhang: Investigation; Methodology; Validation; Conceptualization; Formal analysis; Data curation; Writing-original draft. 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Effects of polymerized whey protein prepared directly from cheese whey as fat replacer on physiochemical, texture, microstructure and sensory properties of low-fat set yogurt. \\u003cem\\u003eLwt-Food Science and Technology\\u0026nbsp;\\u003c/em\\u003e\\u003cstrong\\u003e2019\\u003c/strong\\u003e, \\u003cem\\u003e115\\u003c/em\\u003e, doi:10.1016/j.lwt.2019.108268.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Mozzarella cheese, Pre-acidification, whey proteins, low-fat, texture\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4055582/v2\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4055582/v2\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe aim of the current study was to explore the effect of pre-acidification and whey protein-based fat replacers (CH-4560 and YO-8075) on low-fat Mozzarella cheese (LFC). The color and browning, calcium, meltability, texutre and pizza bake performance of cheeses at 0, 7, 14, 21, 28 days storage was determined, and full-fat cheeses (FFC; milk containing 2.21 g fat/100 g milk) and LFC were manufactured as control. The LFC without fat replacer was harder, less sticky, and had high stretch force and low meltability, as well as poor pizza bake and stretch performance, while these appearance defects were partially corrected via the addition of whey protein concentrates and using pre-acidification. Pre-acidification of cheese milk to pH 5.5 or lower negatively affected the stickiness, and pizza bake, and stretch performance of LFC and LFC with fat replacers. As aging progressed, the meltability, stickiness and stretch performance of Mozzarella cheese improved, whereas the hardness and stretch force of Mozzarella cheese decreased. Our study will provide guidance for the preparation of higher quality low-fat mozzarella cheese.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Effect of pre-acidification and whey proteins on low-fat Mozzarella cheese during storage: physicochemical, textural, and functional properties\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":2,\"date\":\"2024-03-13 19:23:38\",\"doi\":\"10.21203/rs.3.rs-4055582/v2\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}},{\"code\":1,\"date\":\"2024-03-11 06:15:47\",\"doi\":\"10.21203/rs.3.rs-4055582/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"436b1235-a78d-4b70-9bf8-c37ceb867ab8\",\"owner\":[],\"postedDate\":\"March 13th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-03-11T06:15:47+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-03-13 19:23:38\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v2\",\"identity\":\"rs-4055582\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4055582\",\"identity\":\"rs-4055582\",\"version\":[\"v2\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}