**1. Introduction**

The cheese-making process of rennet coagulated cheeses consists in the formation of a three-dimensional network of paracasein, in which fat globules and part of the milk whey are entrapped. The quantity of milk constituents recovered into cheese is strictly dependent on the quality of the milk (casein content, casein micelle structure, and integrity) and conditions of the cheese-making process (for example, pre-acidification of processed milk, type and quantity of rennet, cooking temperature, acidification of the cheese mass), and thus varies depending on the cheese type.

The assessment of the e fficiency of the cheese-making process (ECMP) is crucial for the profitability of cheese-factories. The best way to quantify the ECMP is to measure the recovery of milk constituents into cheese through a mass balance determination. This can be obtained by measuring the quantity of a constituent in the processed milk and the resulting cheese. However, to perform this kind of analysis, it is necessary to also measure the weight of both the processed milk and cheeses. This is not always possible, especially in artisanal cheese factories, as those involved in Parmigiano Reggiano production, where the weight of the processed milk is estimated using a wooden measuring stick with a sensitivity, are not acceptable for research purposes.

Although less reliable than a mass balance, a rough and simple alternative way to estimate the ECMP is to measure the estimated cheese-making losses (ECL). In this method, the estimated loss of

a milk constituent is expressed by the ratio between its concentration in the cheese whey (C-whey) (that remains in the vat after the extraction of the cheese mass) and that in the processed vat milk (V-milk). Consequently, the determination of ECL is easier and faster to perform, since for a single milk constituent it is necessary to only measure its concentration in V-milk and C-whey. No weight of milk, cheese, or whey is needed, and it is not necessary to sample and analyze the cheese. Moreover, to date, the concentration of most milk constituents can be rapidly assessed by applying the mid infrared technology (MIR) [1–3]. The MIR technology is widely used by a lot of laboratories which provide analysis and technological consulting services to dairy farms producing Parmigiano Reggiano cheese. In these laboratories, this kind of analysis is routine, cheap, and certified ISO/IDF. The ECLs have been used in several studies to estimate the e ffect of breed [4], storage conditions [5], and somatic cells [6,7] on e fficiency in Parmigiano Reggiano cheese-making.

Parmigiano Reggiano is a hard, cooked, and long-ripened protected designation of origin (PDO) cheese made from raw milk, following a strict manufacture procedure [8]. In case of raw milk cheese, the quality of milk-in terms of chemical composition and microbial characteristics is one of the main factors influencing the e fficiency of the cheese-making process. Seasonal variations of milk characteristics at the herd level were reported in several studies [9,10]. Significant variations of the chemical and microbial quality of milk employed for Parmigiano Reggiano cheese throughout the year have also been reported [11,12]. These variations could have repercussions on ECLs and curd fines, as reported by Formaggioni et al. [13] and by Franceschi et al. [5]. However, these papers considered a limited number of cheese-making trials and milk traits, since minerals were not taken into account and only two periods of the year were considered. Moreover, in the majority of PDO cheese manufacture, such as Parmigiano Reggiano, where milk standardization and automation of the processes are not implemented, a strong variability in the ECMP is expected among dairy farms [14]. However, the quantification of this variability has never been carried out.

The aim of this research was to evaluate the influence of the season and cheese-factory on ECLs on the quantity of curd particles lost in the whey (curd fines) in Parmigiano Reggiano cheese manufacture carried out in field conditions.

#### **2. Materials and Methods**

#### *2.1. Cheese-Making Process*

Cheeses were produced by the approved method of the Consortium [8,15]. A natural whey starter culture (about 2.5–3 L for every 100 kg V-milk), obtained by the spontaneous acidification of previous day milk whey (C-whey), was added to the V-milk before coagulation. It was then heated to 33 ◦C and clotted in 10–12 min with 2.5 g for every 100 kg V-milk of calf rennet (1:125,000 units). The curd was broken up into small granules (approximately the size of a rice grain) and cooked. During this operation, the temperature was increased in two steps to 55 ◦C within 10–15 min; during this phase the curd was stirred continuously. After cooking the broken-up curd particles, thy were deposited by simple decantation at the bottom of the vat, where they aggregated and blended together spontaneously. In this step, the temperature was 55–53 ◦C and the process lasted 45–60 min. The cheese mass was then removed from the vat, divided into two parts, and placed in special molds called "fascere" for two days. During this period, the cheese wheels were naturally cooled and periodically turned over to allow an homogeneous drying. Furthermore, pH decreased from about 6.0 (at the extraction from the vat) to about 5.1 at the end of the two days. This is related to the activity of thermophilic lactic bacteria added with the natural whey starter, which converts lactose to lactic acid. The cheese wheels were then placed into a saturated brine for a period of 20–25 days. Finally, the cheese entered the ripening phase, a process that lasts about 24 months, and, at the end of the ripening, the cheese wheels resulted in a cylindrical in shape, with a slightly convex side, 22–24 cm high, 40–45 cm diameter, weight 35–36 kg.

#### *2.2. Experimental Design and Sampling Procedure*

The research involved 288 Parmigiano Reggiano cheese-making trials, carried out in 3 cheese factories (CF1, CF2, and CF3), throughout two years.

Briefly, every cheese factory vat (that was filled with the milk of the same herd throughout the two years of the experimental period) was selected (6 vats in CF1, 3 in CF2, and 3 CF3). Each selected vat was sampled once a month, and all the vats of the same cheese factory were sampled on the same day. Each vat always contained the milk from the same farm. From each cheesemaking, samples of V-milk and C-whey were collected, following the International Dairy Federation standard [16]. V-milk samples were collected at the beginning of the cheese making process, before the addition of the natural whey starter culture. C-whey samples were collected after the extraction of the cheese mass and stirring of the whey.

#### *2.3. Analytical Methods*

The following traits were determined or calculated on both V-milk and C-whey. Total N (TN) and non casein N (NCN) were measured on milk and acid whey at pH4.6, respectively, by Kjeldahl, from which the values of crude protein (TN × 6.38/1000) and casein ((TN-NCN) × 6.38/1000) were calculated [7]. Fat content was assessed by the mid infrared method using a FT 6000 (Foss Electric, DK-3400 Hillerød Denmark); dry matter was measured after oven drying at 102 ◦C and ash was measured after muffle calcination at 530 ◦C [17]; total Ca and Mg were determined on a chloridric ash solution by atomic absorption spectrometry (AAS) with a wavelength reading at 422.7 and 285.2 nm, respectively; and total P was assessed on a chloridric ash solution following the colorimetric method [18] with a wavelength reading at 750.0 nm. Titratable acidity was measured only on V-milk by titration of 50 mL of milk with 0.25 N sodium hydroxide with the Soxhlet–Henkel method [7]. Rennet coagulation properties were also measured on V-milk, using Formagraph (Foss Electric, DK-3400 Hillerød Denmark) [4]. The analysis was performed adding 0.2 mL (1:100) of rennet solution (1:19,000; Chr. Hansen, I-20094 Corsico MI, Italy) to milk samples (10 mL). The rennet coagulation properties, milk clotting time, curd firming time, and curd firmness, were measured at 35 ◦C. Milk clotting time is the time from the addition of rennet to the onset of gelation. Curd firming time is the time from the onset of gelation till the signal attains a width of 20 mm. Curd firmness is the width of the signal 30 min after the addition of rennet. To record curd firming time values in milk samples that do not reach a width of 20 mm within 30 min, the analysis was prolonged to 60 min. The curd fines were determined in C-whey by the gravimetric method proposed by van den Berg et al. [19]. In this method, 250 of C-whey were centrifuged at 2000 g for 30 min. The pellet was resuspended in distilled water and filtered on a Whatman 40 filter paper. The filter was dried at 102 ◦C for 2 h and weighed.

ECLs of dry matter, protein, casein, fat, calcium, phosphorus, and magnesium were calculated as follows:

$$\text{ECL} = \text{[C-whey]} \times 100 \text{[V-milk]}$$

where ECL is expressed as percentage; C-whey = concentration in whey, expressed as g/100 g (mg/100 g for Ca, P, Mg); V-milk = concentration in milk, expressed as g/100 g (mg/100 g for Ca, P, Mg).

#### *2.4. Statistical Analysis*

The significance of the differences between seasons and cheese-factories were tested by analysis of variance, using the software for statistical analysis SPSS (IBM SPSS Statics 23, Armonk, New York 10504-1722, NY, USA), according to the following univariate model:

$$\mathbf{Y}\_{\rm ijk} = \mu + \mathbf{S}\_{\rm i} + \mathbf{C}\_{\rm j} + \varepsilon\_{\rm ijk}$$

where Yijk = dependent variable; μ = overall mean; Si = effect of season (i = 1, ... , 4; winter, from January to March; spring, from April to June; summer, from July to September, Autumn, from October to

December); Cj = effect of cheese-factory (j = 1, ... , 3; CF1, CF2, CF3); <sup>ε</sup>ijk = residual error. The Bonferroni post-hoc test was employed to evaluate the significance of the differences between means.

Data were also processed by the Pearson product moment correlation coefficient to measure the degree of linear relationship between milk constituents and ECLs.
