## 2 Materials and Methods
### Methods
#### 2.2.2 Calculation of the Characteristics of the Ultrafiltration Process  
* Volume reduction ratio  
The volume reduction ratio (VRR) was calculated by the following equation:  
\[VRR=\frac{V_{F}}{V_{R}} \tag{1}\]  
where \(V_{F}\) is the volume of the feed buttermilk, dm\({}^{3}\); \(V_{R}\) is the volume of the retenate obtained during ultrafiltration, dm\({}^{3}\).
* Transmembrane pressure  
The transmembrane pressure (\(p\), MPa) was calculated by the following formula:  
\[p=\frac{p_{1}+p_{2}}{2}-p_{3} \tag{2}\]  
where \(p_{1}\) is the feed stream's inlet pressure, Pa; \(p_{2}\) is the concentrate stream pressure, Pa; \(p_{3}\) is the permeate stream pressure, Pa.
* Permeate flux  
The permeate flux (\(J\), L/(m\({}^{2}\) h)) characterizes the volume of permeate generated per unit area of membrane per unit of time. It was calculated according to the following formula:  
\[J=\frac{V}{A\times t} \tag{3}\]  
where \(V\) is the volume of the permeate, L; \(A\) is the membrane area, m\({}^{2}\); \(t\) is the time, h.
* Rejection factor  
The rejection factor (\(R\), %) of the membrane according to the main compounds in buttermilk was calculated using the following formula:  
\[R=\left(1-\frac{C_{p}}{C_{R}}\right)\times 100 \tag{4}\]  
where \(C_{p}\) is the concentration of the compound in the permeate, %; \(C_{R}\) is the concentration of the compound in the retenate, %.
* Concentration factor  
For calculating the concentration factor (\(C_{F}\)) of the main components in buttermilk, the following equation was used:  
\[CF=\frac{C_{R}}{C_{O}} \tag{5}\]  
where \(C_{R}\) is the concentration of the compound present in the retenate, %; \(C_{O}\) is the concentration of the compound present in the feed buttermilk, %.  
* Energy demand For the calculation of the energy demand, it is necessary to know the value of the power required by the pump (\(W_{pump}\), \(W\)). It was estimated using Equation (6) [24; 25; 26].  
\[W_{pump}=(p_{1}\times Q_{feed})/\eta_{pump} \tag{6}\]  
where \(p_{1}\) is the feed stream's inlet pressure without considering the hydraulic resistances because they were negligibly small, Pa; \(Q_{feed}\) is the volumetric flow rate, (m\({}^{3}\)/s); \(\eta_{\mathrm{pump}}\) is the pump efficiency, \(\eta_{\mathrm{pump}}\) = 0.7 according to [22; 26; 27], and type and state of the pump.  
The energy demand per m\({}^{3}\) of permeate (\(E\), kWh/m\({}^{3}\)) was determined [27]:  
\[E=W_{pump}/(J\times A) \tag{7}\]  
where \(J\) is the permeate flux according to Equation (3), m\({}^{3}\)/(h m\({}^{2}\)); \(A\) is the membrane area, m\({}^{2}\).  
2.3 Methods for Analyzing the Composition of the Initial Buttermilk, Retentates and Permeate, and Acidity during Ultrafiltration  
The initial buttermilk, retentates, and permeate were analyzed according to dry matter, protein, fat, ash, and phospholipids content using the following methods:  
* Dry matter content--using ISO 6731:2010 [28];
* Total protein content--by ISO 8968-1:2014 [29];
* Fat content--according to ISO 2446:2008 [30];
* Ash content--according to BS 6154:1974 [31];
* Phospholipid composition--extraction of the buttermilk was made using a mixture of chloroform and methanol (2:1, \(v/v\)) [32]. Precipitation of the phospholipids from the mixture was performed with acetone and then two-dimensional thin-layer chromatography (TLC) was used for the isolation of the individual phospholipids [33]. The identification was made by comparison of the Rf values with authentic standards. The spots of phospholipids were scrapped and mineralized with sulphuric acid and perchloric, 1:1 (\(v/v\)), and the quantification was made spectrophotometrically at 700 nm [34].  
The active and tittatable acidity were determined as follows:  
* Active acidity (pH)--using a pen-type pH meter PH--03 [I] (Hinotek Lab, Ningbo, China);
* Tittatable acidity--according to ISO 6091:2010 | IDF 86:2010 [35].