A New Approach for the Utilization of Technical Egg Albumen Based on Acid–Thermal Coagulation

Abstract

Technical albumen (TA) is liquid waste from egg processing enterprises and occupies a share of 10–15% of the waste. Proteins have the property at the isoelectric point of weakening their repulsive forces. This property is the basis of a TA recovery method using pretreatment to reduce moisture before drying. In this study, we present the results of a TA processing method using two types of citric and phosphoric acids based on thermal–acid coagulation as an alternative to spray drying. By analyzing physicochemical and microbiological indicators, the raw TA and the finished product are described. In this study, the characteristics of raw TA and its final product are presented. TA contains mainly water, fat, and protein, including all essential amino acids, all of the proteins of the egg white, and some of the yolk. Initially, TA is significantly microbiologically contaminated. A better yield was obtained when using citric acid 97.79% instead of phosphoric acid. The final dried egg product from TA has a protein content of 46% and a fat content of 33%. The dried egg products undergo changes in the lipid and protein fraction during storage, but the values remain low TBARS to 4 mg MDA/kg. Microbiological contamination has decreased due to a decrease in water activity to a level that meets the requirements of European legislation for the processing of animal by-products (ABPs) and uses as feed.

1. Introduction

Eggs are an excellent source of nutritional and biologically active substances—proteins and lipids, as well as all essential amino acids. They are defined as the cheapest animal source of protein [1]. Apart from being nutritious, eggs also have functional properties, such as foaming, emulsifying, and gelling ability [2].

Approximately 30% of the egg supply in developed countries is processed into various processed products and not sold as shell eggs [3]. This percentage reaches up to 50% in some countries of the European Union [4]. Egg products are eggs that have been processed and packaged in a convenient form. Some examples of egg products include dried, liquid, or frozen egg white, yolk, or whole egg. These egg products are used in the food industry after heat treatment to remove bacterial contamination [5,6]. Industrialized production of egg products offers economic benefits, but a significant amount of waste is generated [7].

Under the European Commission’s proposals, the circular economy package will aim to minimize waste so that when a product reaches the end of its life, its materials remain in the economy as much as possible. Therefore, what was previously considered “waste” becomes a valuable resource [8]. The 3R principles (reduce, reuse, and recycle) are at the heart of the “circular economy” [9].

Food waste is one of the important factors for the carbon footprint. The consequences of increasing greenhouse gas emissions are climate change, water scarcity, pollution, soil erosion, and a reduction in biodiversity above and below ground. The treatment of food waste through various sustainable methods is of primary importance to reduce the anthropogenic impact on nature [10]. The wastes in the production of egg products are eggshell ~70%, eggshell membrane ~10–15% (solid waste), and adherent (technical) egg white ~10–15% (liquid waste) [11]. It can be estimated that at least 7,894,962 t of egg processing waste is produced annually worldwide, of which 777,000 t is low in the Union [12]. According to European legislation, waste from the processing of eggs with animal by-products is category 3, and the legislation allows it to be processed into feed [13]. Feed materials from egg products are present in the EU feed materials catalog [14].

The conversion of food waste into animal feed is preferable to composting and anaerobic digestion for biogas production or disposal in landfills [15]. Technical albumen (TA) is a viable animal feed after proper processing due to its excellent amino acid profile, energy content, and presence of antimicrobial proteins, but it has received little attention from the industry [16]. The proteins contained in the technical egg white are identical to those of the egg white proteins [12]. An example of protein utilization from this waste product is application in feed for broiler chickens [17] and weanling pigs [18]. Egg waste spray drying technology is a common method of producing animal feed from this type of waste. According to Banožić et al., spray drying is used to process egg by-products into feed, but the biggest disadvantage of this type of processing is the high investment costs [19].

In our previous research, we found that acid–thermal coagulation is a possible alternative way to treat liquid waste from egg processing. In our preliminary study, we used four different types of acids. From them, the most suitable acids for pH correction are citric acid (CA) and phosphoric acid (PA), which showed the best values of the production indicators yield and pressing efficiency [20]. In this study, we characterized the raw TA and the final product from it. We evaluate the most suitable pretreatment to reduce moisture before drying in order to reduce the cost and heat impact on dry egg products. The initial characteristics of produced dried egg products from TA will be evaluated, as well as their changes during storage. We describe and compare the obtained characteristics of the finished product

2. Materials and Methods

2.1. Materials

The technical egg white samples were supplied by the company “OVO-BUL” Ltd., (Town of Pleven, Republic of Bulgaria) a registered manufacturer of pasteurized egg products. TA was obtained from the shells of broken eggs, from the company’s usual production of egg products for human consumption, after centrifugation in a SANOVO SEC 360 centrifuge. The amount provided was separated in a stream up to 5 min after receiving the product in polyethylene bags. Technical packaging of the protein was frozen (TA batch 1) or pasteurized in a water bath at 50 °C for 20 min (TA batch 2). Until performing the analysis with TA, it was transported and stored at a temperature of 0–4 °C for no more than 24 h.

2.2. Methods

2.2.1. Raw Materials

TA Extraction

The TA was processed according to the method of Saraliev et al. [20] with modifications. Citric and phosphoric acid were used for pH correction. Part of the acidified samples were additionally treated with n-hexane to extract the fats. Separation of fats from the samples treated with n-hexane and treated with citric acid (CADF) and phosphoric acid (PADF) is carried out in function before the coagulation process. The obtained coagulates are pressed to evaluate the pressing efficacy, followed by drying in a hot rack oven at 80 °C for 6 h, according to COMMISSION REGULATION (EU) No. 142/2011 [21].

Proximate Composition

Protein content was determined by the Kjeldahl AOAC method [22]—BUCHI EasyKjel (BÜCHI Labortechnik AG, Flawil, Switzerland) equipped with an EcoTitrator (Metrohm AG, Herisau, Switzerland) with a nitrogen–protein conversion factor = 6.25. The amount of fat was determined by extraction with diethyl ether in a Soxhlet apparatus [23]. Moisture content was determined by drying to constant weight at 104–105 °C [24]. The total ash content was determined by burning the samples in a muffle furnace at 400–600 °C [25].

Protein Separation and Identification

Diluted sample was mixed with a Laemlli Sample Buffer (Sigma-Aldrich, Co., St. Louis, MI, USA) in a 3:1 proportion, boiled, cooled, centrifuged, and injected into 10% polyacrylamide gels [26]. SDS-PAGE gels were at a constant voltage mode (200 V) and were used in an Omni PAGA Electrophoresis System (Cleaver Scientific Ltd., Rugby, UK) [27]. A protein marker was used as the Precision Plus Protein Standards (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Amino Acid Profiling

Initially, the samples were subjected to acid hydrolysis with 6N HCl for 24 h. The hydrolysates were dried at 60 °C and subsequently dissolved in 20 µM HCl. The derivatization of the samples was performed using a kit for the determination of amino acids—ACCQ-FLUOR REAGENT KIT (Waters Millipore, Bedford, VA, USA). The separation of the derivatives was carried out by high-performance liquid chromatography at a temperature of 37 °C and a wavelength of 244 nm [28]. For this purpose, two liquid phases were used—ACCQ-FLUOR Buffer (Waters Millipore, Bedford, VA, USA) and 60% acetonitrile (Sigma-Aldrich, Burlington, MA, USA) in ratio, time, and flow rate according to

Table 1. Data on parameters used in amino acid profile determination.

Time, min	Buffer—A, %	AN (60%), %	Flow, mL/min
0	100	0	0.9
0.5	98	2	0.9
18	93	7	0.9
22	90	10	0.9
33	67	33	0.8
34	65	35	0.8
36	50	50	0.8
38	0	100	0.8
40	100	0	0.8

. A liquid chromatography system was used, Hitachi LaChrom Elite^® HPLC System (Hitachi High Technologies America, Inc., Schaumburg, IL, USA) equipped with a diode array detector (DAD, L-2455) and EZChrom Elite™ software version 3.2.1 (Scientific Software, Inc. © Agilent 2005–2007, Santa Clara, CA, USA)and a c18 reverse phase column (Waters Millipore, Bedford, VA, USA).

Alkaline hydrolysis was performed by mixing 100 mg of dry sample and 5 mL of 4 N NaOH in a glass ampoule sealed in an inert medium. Hydrolysis was for 4 h at 104 °C, followed by cooling in ice to room temperature. The solution was neutralized to a pH of about 7.0 using 12 N HCl and diluted with borate buffer pH 9.0 to a volume of 25 mL [29]. The spectrophotometric determination of the amount of tryptophan was performed according to the method described by Opienska-Blauth et al. [30].

2.2.2. Physicochemical Characteristics

pH Determination

pH determination is measured electropotentiometrically with a pH meter Microsyst MS 2004 (Microsyst, Plovdiv, Bulgaria), equipped with a temperature and combined pH electrode Sensorex Combination Recorder S 450 CD (Sensorex pH Electrode Station, Garden Grove, CA, USA), which was pre-calibrated with certified buffer solutions of pH 4.04 and 6.86 [31].

Color Characteristic Determination

The color is determined according to the CIE Lab system, using a Konica Minolta CR-410 calorimeter (Konica Minolta Holding, Inc., Ramsey, NJ, USA) equipped with standard observer 2° at an aperture = 8 mm and light source D65. The color brightness (L*), red (a*), and yellow (b*) component parameters of the color were measured [32].

Water Binding Capacity (WBC)

The procedure was performed according to the method described by Sze et al. [33] with modifications. Briefly, 0.5 g of dry egg white sample was mixed with 10 mL of distilled water in a centrifuge tube. The tubes were shaken vigorously for 30 s, followed by centrifugation at 3500 rpm for 25 min. The tubes were placed on filter paper for 1 h to separate the supernatant. The water binding capacity of dry egg white products is calculated according to the following Equation (2):

$$\mathrm{WBC}={\displaystyle \frac{\mathrm{final} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{sample}, \mathrm{g}}{\mathrm{initial} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{sample}, \mathrm{g}}}\times 100,\%$$

(1)

Water Activity (a_W)

The water activity of the samples was determined using a Novasina AG CH-8853 aw meter (Novasina AG, Zurich, Switzerland) at 20 °C. Initially, the sample is homogenized and placed in the sample holder (representing a small plastic Petri dish), filling a maximum of 2/3 of its volume [31].

2.2.3. Microbiological Status

Sample suspension preparation and decimal dilutions were performed according to ISO 6887-4:2017 [34]. The microbiological status is represented by the total plate count (TPC), the number of coliforms, the number of E. coli, and the number of members of the family. Enterobacteriaceae, as well as Salmonella spp., following the procedures outlined in ISO 4833-1:2013/Amd 1:2022 [35].

2.2.4. Production Parameters

Pressing Efficiency (PE%)

The production parameter describes the product’s ability to release liquid by applying pressure to it. It represents the ratio of the amount of liquid released during pressing to the initial amount of product expressed as a percentage. A larger value of EP indicates a lower amount of water in the coagulant and a better efficiency of the pressing process.

$$\mathit{P}\mathit{E}={\displaystyle \frac{\mathrm{initial} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{sample}, \mathrm{g}-\mathrm{pressed} \mathrm{material}, \mathrm{g}}{\mathrm{initial} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{sample}, \mathrm{g}}}\times 100,\%$$

(2)

Yield (Y)

Yield is an indicator of the amount of final product that can be obtained from a unit of raw product. TA is a raw material that has a different amount of dry matter. The yield depends on the initial amount of dry matter in the raw product, so in our research, we present the yield as a percentage of the theoretically possible.

$$\mathit{Y}={\displaystyle \frac{\mathrm{final} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{sample}, \mathrm{g}}{\mathrm{initial} \mathrm{mass} \mathrm{of} \mathrm{the} \mathrm{sample}, \mathrm{g}.\mathrm{DCrp}/100}}\times 100,\%$$

(3)

DCrp—Dry content of raw product, %

2.2.5. Hydrolytic and Oxidative Changes

Oxidative Changes in Lipid Fraction

The secondary products of lipid oxidation are expressed by the indicator thiobarbituric reactive substances (TBARS) and determined spectrophotometrically according to the method of Botsoglu et al. [36], with modifications. Initially, 5 g of dried egg product from technical albumen is mixed with 50 mL 0.9% NaCl, shaken, and let to rest for 10 min. Secondly, 50 mL 10% trichloroacetic acid is added to precipitate the extracted proteins. The solution is filtered, and 4 mL of the clear extract is mixed with 1 mL of 1% 2-thiobarbituric acid and incubated in water bath at 70 °C for 30 min. The absorption is measured at 532 nm using a double-beam UV–VIS spectrophotometer Camspec M550 Spectronic CamSpec Ltd., Leeds, UK).

Hydrolytic and Oxidative Changes in Protein Fraction

The amount of soluble protein in dried egg products was determined by the method of Lowry et al. [37] with some modifications. Briefly, dry egg white products are mixed in a ratio of 1:20 with a phosphate buffer of pH 7.3 and left overnight in a refrigerator. The solutions are filtered, and if necessary, the filtrate is diluted. Mix 1 mL of filtrate and 5 mL of reagent C in a tube. The samples are left for 10 min at room temperature, after which 0.5 mL of reagent D is added. The absorbance is measured after 30 min at 750 nm against a blank containing water instead of filtrate. The amount of soluble protein is presented as mg bovine serum albumin (BSA)/mL. Reagent A—2% Na₂CO₃ in 0.1 N NaOH; Reagent B—0.5% CuSO₄ in 1% sodium citrate; Reagent C—Mixture of Reagent A Reagent B in 50:1 ratio; Reagent D—Folin’s reagent (Folin–Ciocalteu).

Hydrolysis and oxidation in the protein fraction were determined spectrophotometrically by the amount of free amine nitrogen (FAN) and protein carbonyls content (PC) by the methods of Vassilev et al. [38] and Mercier et al. [39].

2.2.6. Data Processing

One-way ANOVA was used to determine whether the variability of the results was due to chance or to the factor in the analysis—significant variation between both batches of technical albumen. Two-way ANOVA was used to determine the significance of the factors influencing the parameters of the dried egg products from technical albumen—type of acid used and time of storage. Results are presented as mean ± SEM at a significance level of α = 0.05. The procedure was executed via Analysis ToolPak of Microsoft Excel Office Professional Plus 2016.

3. Results

3.1. Raw Materials Characterization

The proximate composition of both used batches of technical protein is presented in Figure 1. The main component of technical protein is water. Protein and ash contents were similar in both samples and quantitatively similar to technical albumen. The fat content found is high and uncharacteristic for egg whites. The amount of fat differed significantly between the two samples, 5.3% in TA1 and 7.2% in TA2 (p ≤ 0.05).

The protein separation and identification by SDS-PAGE showed that the protein profile of the TAare presented all of the proteins found in egg white (Figure 2). For example, we determined the presence of Ovotransferin with 77.7 kDa molecular weight, Ovoglobulin 49 kDa, Ovoalbumin 45 kDa, and Lysozyme 14.3 kDa. In the protein profile of the technical albumen, proteins like g-livetin—203 kDA, Apovitellin 3 + 4—110 kDa, Apovitellin Vb—93 kDa, Apovitellin V—85 kDa, Apovitellin IV—68 kDa, α-livetin 55 kDa and Apovitellin 8–31 kDa were also identified. No significant difference (p ≥ 0.05) was established between both batches of technical albumen.

The amino acid composition of the two batches in

Table 2. Aminoacidic profile of the technical albumen.

mg/mL	TA1	TA2	p-Value
Aspartic acid	6.63 a ± 0.39	7.16 a ± 0.41	0.827886
Serine	4.05 a ± 0.43	4.37 a ± 0.45	0.712457
Glutamic acid	3.62 a ± 0.39	3.91 a ± 0.40	0.844731
Glycine	1.67 a ± 0.24	1.80 a ± 0.25	0.75009
Histidine	2.47 a ± 0.33	2.66 a ± 0.34	0.842366
Arginine	13.18 a ± 0.19	14.24 a ± 0.19	0.802974
Threonine	3.06 a ± 0.44	3.30 a ± 0.45	0.61783
Alanine	7.56 a ± 0.47	8.16 a ± 0.48	0.80066
Proline	3.77 a ± 0.42	4.07 a ± 0.43	0.700507
Cysteine	0.08 a ± 0.08	0.09 a ± 0.08	0.823836
Tyrosine	3.17 a ± 0.54	3.42 a ± 0.56	0.588348
Valine	2.56 a ± 0.32	2.76 a ± 0.33	0.732944
Methionine	1.62 a ± 0.25	1.75 a ± 0.26	0.722243
Lysine	6.44 a ± 0.38	6.95 a ± 0.39	0.793566
Isoleucine	2.18 a ± 0.29	2.35 a ± 0.31	0.727851
Leucine	3.63 a ± 0.45	3.92 a ± 0.47	0.687175
Phenylalanine	1.91 a ± 0.45	2.06 a ± 0.47	0.380355
Tryptophan	3.00 ± 0.21	3.55 a ± 0.19	0.935071

Results are presented as means ± SEM; superscripts indicate significant (p ≤ 0.05) differences between both batches of technical albumen for each parameter separately.

presents that all essential amino acids are found in both batches of technical albumen. Similar to the protein profile, the amino acid composition is identical in both batches of technical albumen.

The physicochemical analysis of the two batches of TA found that the pH values ranged from 7.7 to 7.9 (

Table 3. Color characteristics of technical protein.

	TA1	TA2	p-Value
pH value	7.91 a ± 0.02	7.73 b ± 0.01	0.0002
L*	66.50 a ± 0.05	65.70 b ± 0.17	0.0008
a*	10.72 b ± 0.03	11.62 a ± 0.03	2.41446 × 10−6
b*	39.31 a ± 0.12	36.36 b ± 0.12	7.33241 × 10−6
C	40.75 a ± 0.11	38.17 b ± 0.11	8.1569 × 10−6
h	74.76 a ± 0.07	72.29 b ± 0.08	2.5518 × 10−6

Results are presented as means ± SEM; superscripts indicate significant (p ≤ 0.05) differences between both batches of technical albumen for each parameter separately.

). Such values are completely characteristic of egg white, which is why it is considered the most alkaline biological fluid [32].

The evaluation of the instrumental color characteristics of the two batches of the technical egg white finds a significant difference (p ≤ 0.05). The lightness of the color (L*), yellowness (b*), saturation index (C), and hue angle (h) was greater in the first batch (TA1), while only the redness of the color (a*) was higher in the second batch (TA2).

No significant differences (p ≥ 0.05) were found while comparing the total plate count (TPC) of both batches of technical albumen (

Table 4. Microbiological status of the technical albumen.

CFU/g	TA1	TA2	p-Value
TPC	6.90 a ± 0.06	7.13 a ± 0.20	0.129065
Coliforms	4.70 b ± 0.16	5.35 a ± 0.30	0.029619
Enterobacteriaceae	20 b	60 a	0.003448
Salmonella spp.	N.F.	N.F.	-

Results are presented as means ± SEM; superscripts indicate significant (p ≤ 0.05) differences between both batches of technical albumen for each parameter separately. N.F.—Not Found.

). Meanwhile, the count of coliforms and representatives of the Enterobacteriaceae family was greater in the second batch (TA2). In both batches, there was no presence of Salmonella spp.

3.2. Processing of the Technical Albumen

The method used for the treatment of the waste product is based on coagulation at the isoelectric point of the proteins of the technical albumen and subsequent pressing in order to reduce moisture. During pressing, a liquid (serum) and a solid phase (coagulate) are formed. The amount of liquid separated determines the efficiency of pressing (PE%). The coagulate is dried, and its amount determines the yield. Larger amounts of serum mean more efficient pressing and subsequent drying but also a reduction in yield because there is a small amount of dry matter in the serum, mostly remaining non-coagulated proteins.

The highest values of yield were obtained in the samples with CA 5.1 97.79% and CADF 5.0 94.79% (Figure 3). They obtained low PE% values of 30.53% and 26.67%, respectively. The sample with the highest PE% value was CADF 5.1, with a yield value of 35.57%. The results show that it is necessary to make a compromise between the two technological parameters. Among the samples using PA, the best yield was obtained from the sample at pH 4.9–91.41% and from the samples with CA pH 5.1.

3.3. Characterization and Changes During Storage of Dried Technical Albumen Products

3.3.1. Proximate Composition and Aminoacidic Profile

The proximate composition of the dried technical albumen products (DTAP) is presented in Figure 4. Drying the coagulate leads to evaporation of water and concentration of the ingredients, while the ratio between the chemical ingredients is preserved. Proteins are in the largest amount in all samples, and this share is the largest in the sample prepared solely with citric acid (CA), followed by those defatted with n-hexane and acidified by phosphoric acid (PADF). Fat ranged from 32.69% in CA to 33.56% in CADF samples. Pre-defatting of the samples does not reduce the fat in the final product.

The results present the influence of the two acids used for the preparation of the dry egg products from technical protein and also their combination with n-hexane. The amino acid composition of dry egg white products is represented by the 18 most common amino acids in egg white (

Table 5. Aminoacidic profile of the dried egg products from technical albumen.

mg/g Protein	CA	CADF	PA	PADF	p-Value
Aspartic acid	74.34 b ± 0.52	109.65 a ± 2.02	51.46 c ± 0.51	38.78 d ± 1.74	0.000295
Serine	48.00 b ± 0.66	68.04 a ± 1.72	29.74 c ± 0.88	33.70 c ± 1.41	0.00355
Glutamic acid	70.79 a ± 1.00	88.38 a ± 1.83	65.56 a ± 0.72	70.34 a ± 1.26	0.183027
Glycine	30.19 b ± 0.99	34.98 a ± 1.19	24.34 c ± 0.41	17.92 d ± 1.25	0.0073
Histidine	57.55 a ± 0.55	61.24 a ± 0.83	48.88 a ± 1.22	47.02 a ± 1.00	0.068277
Arginine	298.35 a ± 1.38	309.28 a ± 1.60	218.83 b ± 3.31	22.21 c ± 0.24	0.018412
Threonine	63.99 a ± 2.16	78.63 a ± 2.43	41.64 a ± 1.22	52.77 a ± 1.15	0.063141
Alanine	154.59 a ± 3.63	212.98 a ± 4.59	121.46 a ± 2.66	135.03 a ± 2.51	0.185893
Proline	79.51 b ± 1.31	98.50 a ± 2.26	59.70 c ± 1.86	44.13 d ± 1.56	0.003679
Cysteine	2.49 a ± 0.27	1.89 a ± 0.61	3.70 a ± 0.35	1.88 a ± 0.66	0.087418
Tyrosine	69.85 a ± 1.97	72.51 a ± 2.12	31.48 b ± 0.62	38.40 b ± 0.72	0.003063
Valine	31.31 b ± 0.89	46.36 a ± 1.59	26.28 c ± 0.83	21.03 d ± 0.24	0.001462
Methionine	30.88 b ± 1.08	38.90 a ± 1.47	19.57 c ± 0.70	23.14 c ± 1.25	0.009944
Lysine	143.08 b ± 2.53	185.54 a ± 3.51	82.18 c ± 1.44	93.25 c ± 1.00	0.004116
Isoleucine	27.67 b ± 0.90	42.11 a ± 1.55	22.03 c ± 0.37	19.34 c ± 0.42	0.00144
Leucine	34.21 b ± 1.33	63.87 a ± 2.22	20.61 c ± 0.64	16.74 c ± 0.26	0.001309
Phenylalanine	22.95 b ± 1.45	36.64 a ± 1.84	8.73 c ± 0.40	10.29 c ± 0.45	0.005354
Tryptophan	20.29 a ± 0.81	19.62 a ± 0.30	19.41 a ± 0.40	22.21 a ± 0.24	0.086523

Results are presented as means ± SEM; superscripts indicate significant (p ≤ 0.05) differences between the dried technical albumen products for each parameter separately.

).

3.3.2. Changes in Physicochemical Properties During Storage

Different types of processing of technical egg white affect the pH value (

Table 6. Changes in physicochemical properties of the dried egg products from technical albumen during storage.

	CA	CADF	PA	PADF	p-Value
24 h after production
pH value	5.32 a,x ± 0.01	5.15 c,x ± 0.01	5.22 b,x ± 0.01	5.12 d,x ± 0.02	3.77 × 10−8
L*	80.72 a,x ± 1.23	78.00 b,x ± 0.25	80.88 a,x ± 1.33	80.15 a,x ± 1.00	0.0315
a*	4.17 b,x ± 0.41	5.96 a,x ± 0.12	4.09 b,x ± 0.42	4.41 b,x ± 0.38	0.0374
b*	39.49 b,x ± 0.54	43.81 a,x ± 0.33	39.45 b,x ± 0.60	39.64 b,x ± 0.59	0.0165
WBC, %	10.73 a,y ± 0.95	4.86 d,y ± 0.12	7.93 c,y ± 0.37	9.47 b,y ± 0.22	0.0134
aW	0.541 b,x ± 0.004	0.655 a,x ± 0.001	0.500 c,x ± 0.010	0.512 c,x ± 0.004	3.44 × 10−9
1 month after production
pH value	5.27 a,x ± 0.04	5.17 b,x ± 0.04	5.25 a,x ± 0.06	5.11 c,x ± 0.01	0.0008
L*	81.04 a,x ± 0.09	78.62 c,x ± 0.13	80.33 b,x ± 0.15	80.35 b,x ± 0.14	0.9452
a*	3.52 d,y ± 0.08	5.27 a,y ± 0.09	4.13 b,x ± 0.09	3.82 c,y ± 0.09	0.3968
b*	37.69 c,y ± 0.14	41.83 a,y ± 0.17	39.81 b,x ± 0.38	37.92 c,y ± 0.22	0.2705
WBC, %	16.40 c,x ± 0.58	20.59 a,x ± 0.41	18.61 b,x ± 1.00	18.69 b,x ± 0.46	9.18 × 10−9
aW	0.461 b,y ± 0.001	0.464 a,y ± 0.002	0.462 a,y ± 0.002	0.463 a,y ± 0.002	0.0002

Results are presented as means ± SEM; superscripts ^a,b,c,d indicate significant (p ≤ 0.05) differences between the dried technical albumen products for each parameter separately, while superscripts ^x,y indicate significant (p ≤ 0.05) differences between time periods.

). The highest (p ≤ 0.05) initial pH values were found in samples treated with citric acid (CA and CADF). The samples with PC and PADF are characterized by significantly lower (p ≤ 0.05) pH values compared to CA, by 3.2 and 3.8%, respectively.

Different types of processing of technical egg white affect the pH value (Table 6). The highest (p ≤ 0.05) initial pH values were found in samples treated with citric acid (CA and CADF). The samples with PA and PADF are characterized by significantly lower (p ≤ 0.05) pH values compared to CA, by 3.2 and 3.8%, respectively. When comparing the pH value of the four samples, statistically insignificant differences (p ≥ 0.05) were found after one month of storage compared to day 1. The trend from day 1 was also maintained during the storage period, with the lowest (p ≤ 0.05) pH value recorded in the PADF sample, followed by the FA sample. At the same time, the pH values of samples CA and CADF stabilize and are statistically indistinguishable (p ≥ 0.05).

The color brightness L* of dry egg products from technical white, determined on the first day of storage, had the highest values in the samples acidified with citric acid (Table 6). The lowest value of the color brightness index L* (p ≤ 0.05) was found in the PA sample obtained by acidifying the technical protein with phosphoric acid. The L* color brightness values of the dry egg products obtained from the technical albumen after one month of storage were statistically indistinguishable (p ≥ 0.05) from previous studies. Again, the highest values were recorded in the CA sample, correspondingly acidified with citric acid. The samples CADF and PADF, respectively, skimmed egg products acidified with citric and phosphoric acid, were statistically insignificantly different from each other (p ≥ 0.05). The lowest value of color brightness L* (p ≤ 0.05) was found in the FC sample obtained by acidifying the technical protein with phosphoric acid.

The highest (p ≤ 0.05) initial water binding capacity (WBC) was reported for sample CA, followed by sample PA and CADF (Table 6). The PA sample is characterized by the weakest (p ≤ 0.05) water binding capacity, which is more than two times weaker than that found for CA. In a native system, there is a direct relationship between pH parameters and WBC, as at certain pH values, proteins can denature or aggregate, affecting the WBC of the given system.

After one month of storage, an increase (p ≤ 0.05) was found in the water binding capacity of dry technical egg white products compared to the initial values. During this period, the value of the PA sample was the highest (p ≤ 0.05), followed by the defatted samples. The latter are statistically the same (p ≥ 0.05). In contrast to the initial values, the CA sample had the best WBC compared to the other studied samples after one month of storage (p ≤ 0.05).

The water activity in the samples, after one month of storage, significantly decreased (p ≤ 0.05) compared to the values reported on the first day. Values of the studied parameter stabilize around 0.462, the differences being statistically insignificant (p ≥ 0.05). The lower values are most likely a result of their relationship with water content.

3.3.3. Changes in Protein Fraction During Storage

No differences (p ≥ 0.05) were found in the amount of soluble protein between samples CA, CADF, and PADF on the first day of their storage (

Table 7. Changes in protein fraction of the dried egg products from technical albumen during storage.

	CA	CADF	PA	PADF	p-Value
24 h after production
SP, mg BSA/g	211.79 a,x ± 0.58	163.53 b,x ± 0.48	218.85 a,x ± 0.85	204.89 a,x ± 0.67	0.001165
FAN, mg alanine/g	117.55 a,x ± 0.25	118.69 a,x ± 0.38	111.24 a,x ± 0.28	118.97 a,x ± 0.49	0.176337
PC, nmol/g protein	0.35 a,y ± 0.04	0.40 a,x ± 0.08	0.26 a,y ± 0.13	0.43 a,y ± 0.08	0.017052
1 month after production
SP, mg BSA/g	166.85 b,y ± 0.70	161.23 b,x ± 0.65	182.85 a,y ± 0.67	150.60 c,y ± 0.85	0.0007
FAN, mg alanine/g	102.72 a,y ± 0.41	102.33 a,y ± 0.45	110.24 a,x ± 0.31	98.78 a,y ± 0.38	3.28 × 10−6
PC, nmol/g protein	0.73 a,x ± 0.16	0.33 b,x ± 0.07	0.80 a,x ± 0.14	0.73 a,x ± 0.16	0.0084

Results are presented as means ± SEM; superscripts ^a,b,c indicate significant (p ≤ 0.05) differences between the dried technical albumen products for each parameter separately, while superscripts ^x,y indicate significant (p ≤ 0.05) differences between time periods. SP—soluble protein; FAN—free amino nitrogen; PC—protein carbonyls.

). In contrast to the other three samples, the PA sample was characterized by about 22% less (p ≤ 0.05) soluble protein. In the one-month storage period, only the PA sample kept stable (p ≥ 0.05) values of the studied parameter. For the same period, a significant decrease (p ≤ 0.05) in soluble protein was found in CA, CADF, and PADF samples. The observed decrease ranged from 16 to 26.5% (p ≤ 0.05) among the tested samples. During the studied period, it was reported that sample CADF was characterized by the highest (p ≤ 0.05) amount of soluble protein, followed by samples LA and PA.

The amount of free amine nitrogen in the four investigated samples did not differ significantly (p ≥ 0.05) during the 1st day of storage. Accordingly, it can be assumed that the different types of processing do not have a direct effect on the hydrolytic changes in dry egg white products. The amount of free amine nitrogen in all investigated samples significantly decreased (p ≤ 0.05) during the one-month storage compared to its initial values. A tendency established at the beginning of the experiment that the different types of treatment do not have an effect (p ≥ 0.05) on the amount of free amine nitrogen was also noted after one month of storage of the samples.

At the beginning of the experiment, the amount of protein carbonyls in the four samples did not differ statistically significantly (p ≥ 0.05).

After one month of storage of the dry egg products, an increase (p ≤ 0.05) was found in the amount of protein carbonyls compared to the initial levels. Insignificant oxidative changes (p ≥ 0.05) were found in the PA sample, which was also characterized by the least protein (p ≤ 0.05). A 2- to 3-fold (p ≤ 0.05) increase in the amounts of protein carbonyls was found over day 1. Sample PADF was characterized by the largest reported value (p ≤ 0.05), which was almost four times greater than that for sample PA.

3.3.4. Changes in Lipid Fraction During Storage

It was found that the amount of the end products of lipid oxidation expressed by the indicator thiobarbiturate number (TBARS) presented in Figure 5 on the 1st day of storage was the least (p ≤ 0.05) in sample CA. Samples PA and CADF are characterized by significantly higher (p ≤ 0.05) values of TBARS compared to CA, with 60.5 and 57.5%, respectively. During the same studied period, the largest amount of the end products of lipid oxidation was found in the PADF sample, which was 3.8 times greater than CA.

After one month of storage, only thiobarbiturate number values in the CADF sample remained stable (p ≤ 0.05) compared to the baseline. A significant increase (p ≤ 0.05) in thiobarbiturate number values was found in the remaining three examined dry egg products from TA, compared to those recorded on the first day. The observed increases in TBARS ranged from 1.5-fold to about 4-fold among the samples examined. Phosphoric acid (PA)-treated non-defatted egg products had the highest (p ≤ 0.05) TBARS values along with PADF, the difference between them being statistically insignificant (p ≥ 0.05). In descending order, the CA sample is arranged, and the lowest (p ≤ 0.05) value of TBC was found in the CADF sample.

3.3.5. Changes in Microbial Status During Storage

A similar initial (p ≥ 0.05) total number of mesophilic aerobic microorganisms, expressed by the index total microbial number mentioned in

Table 8. Changes in microbiological status of the dried egg products from technical albumen during storage.

CFU/g	CA	CADF	PA	PADF	p-Value/ Significance F
24 h after production
TPC	4.92 a,x ± 0.47	4.73 a,x ± 1.33	4.81 a,x ± 0.39	5.37 a,x ± 0.35	0.808425
Coliforms	4.00 a ± 1.00	3.00 b ± 0.420	4.15 a ± 1.15	3.83 a ± 0.62	0.768197
E. coli	N.F.	N.F.	N.F.	N.F.	-
Enterobacteriaceae	<10	<10	<10	<10	-
Salmonella spp.	N.F.	N.F.	N.F.	N.F.	-
1 month after production
TPC	3.96 a,x ± 0.41	4.65 a,x ± 0.37	4.41 a,x ± 0.37	4.40 a,x ± 0.38	0.1670
Coliforms	N.F.	N.F.	N.F.	N.F.
E. coli	N.F.	N.F.	N.F.	N.F.	-
Enterobacteriaceae	<10	<10	<10	<10	-
Salmonella spp.	N.F.	N.F.	N.F.	N.F.	-

Results are presented as means ± SEM; superscripts ^a,b indicate significant (p ≤ 0.05) differences between the dried technical albumen products for each parameter separately, while superscripts ^x indicate significant (p ≤ 0.05) differences between time periods. N.F.—Not Found.

, was found in samples CA, PA, and CADF. In sample PADF, the highest (p ≤ 0.05) degree of microbial insemination was evaluated.

After a month of storage, a difference was found in the PADF sample, whose number of mesophilic aerobic microorganisms decreased (p ≤ 0.05) compared to the initial one. The remaining three samples remained stable (p ≥ 0.05) relative to their baseline values. At the same time, the difference between the four samples is statistically insignificant (p ≥ 0.05).

The detected number of coliforms was the lowest in sample PA (p ≤ 0.05). CA and CADF samples, as well as PADF, had statistically insignificant differences (p ≥ 0.05). No presence of E. coli was detected in all the tested samples. Microbiological analysis of the four samples stored for one month did not detect the presence of both coliforms and E. coli. This is probably due to the low water activity resulting from moisture loss during storage.

It was found that in all investigated samples, the number of Enterobacteriaceae microorganisms was below 10 CFU/g (p ≥ 0.05), both at the beginning of the experiment and at the 1st month of storage (Table 8).

The examination of the dry egg products from technical protein did not detect the presence of Salmonella spp. for the entire period of the experiment.

4. Discussion

Waste from egg processing plants constitutes a significant amount of the food processing industry. The second in quantity of these wastes is liquid TA waste [12]. Egg waste products are animal by-products (ABP) category 3. The management of these products, due to safety reasons, is strictly regulated by European legislation. The ABP regulations set out rules for the collection, transport, storage, handling, processing and marketing, import, export, and transit of crude ABPs and products derived from them, which are among the strictest in the world. Feed costs are the largest cost of farming livestock in Europe, mainly due to the need to use imported protein ingredients (soybean and fishmeal), and can be reduced by investing in other protein-rich sources. Also, egg products circumvent the prohibitions of Regulation 999/2001 [40] on feeding farm animals with feed containing sources of animal origin. Feed ingredients from egg waste products can be fed to all categories of animals, including ruminants [41]. European legislation allows the use of ABP category 3 as animal feed, and TA is particularly suitable due to its chemical composition (Figure 1). It contains a significant amount of water but also nutrients—mainly proteins and fats.

It is known that all the fat in eggs is found in the yolk. During the mechanical breaking of eggs in egg processing plants, the vitelline membrane of the yolk is broken, and part of it falls into the TA. The different amounts of fat in the samples may be due to the different eggs used as raw material. Other factors that have an influence are the speed of breaking the temperature of the eggs. The fat content of this product varies and cannot be predicted. The established amino acid composition confirms the statement that the nutritional completeness of the TA is no less than that of egg white intended for human consumption [42]. The pollution of TA with yolk during processing is the most logical explanation for the increase in the yellow component of the color (b*).

In the context of the circular economy, this waste should be considered as raw material for animal feed after appropriate processing meets the feed criteria [8]. The raw TA has an unstable composition and is microbiologically inoculated but does not contain the pathogenic Salmonella spp. The high microbial contamination is a consequence of the method of obtaining this product by crushing the eggshells. For this reason, ref. [43] prohibits its use for human consumption. Our microbiological results of the raw product for TPC, coliforms, and Enterobacteriaceae indicators confirm that TA does not meet the criteria and should be used after heat treatment (Table 3). In order for ABPs to be used, they must first be processed to meet the microbiological criteria specified in Regulation 142/2011 [21]. A method based on acid–thermal coagulation is suitable for processing this material. Heat treatment and subsequent drying of the product ensure a reduction of the microbial population. In the final product, after a month of storage, bacteria from the family Enterobacteriaceae, coliforms, and E. coli are not detected, and TPC has decreased to an acceptable level (Table 7).

Our results show that in the pretreatment before drying, the product is partially dehydrated. In the pressing process, the product loses up to 45% of its water content. This treatment facilitates drying because it starts from a lower moisture level. In addition, due to the low energy costs, the drying time and the impact of high temperatures on the product are reduced. This, in turn, reduces the formation of unwanted compounds that can be monitored by measuring the color characteristics.

The best yields are obtained using CA 97.79%; this value indicates that the dry matter losses during the whole processing process for this sample are about 2.2%. The dry matter losses in this method of processing liquid egg processing waste are in the serum obtained during pressing. This serum contains varying amounts of non-coagulated soluble proteins and fats. Preliminary degreasing of the product does not lead to improvement of the production parameters. The dry end product contains over 46% protein and about 33% fat. These values are close to the stated protein and fat content of dry egg feed materials of 50.9% and 31.1%, respectively [44]. Also close are the values for protein and fat processed differently reported in the study (Figure 4). It can be concluded that the raw material and the type of processing do not have a significant impact on the fat and protein content of the final product. Norberg et al. and Song et al. also investigated the amino acid profile of spray-dried ABPs from eggs [44,45]. The results of the authors for the presence of amino acids in these products differ significantly from each other. Our repeated results for amino acid content also differed significantly for some amino acids. With these data, we can state that the amino acid profile of dry egg products is different depending on the raw material and the method of processing. The non-constant amino acid composition of the product can create difficulties in its dosing in feed production.

Our final products had low moisture and low water activity. These low values contribute to the easy storage and transport of the obtained product [33]. The increase in water binding capacity during the storage period is a potential result due to the observed moisture loss of the investigated samples. All samples were found to be characterized by about 3–4% moisture at the end of storage, which was significantly lower (p ≤ 0.05) than the initial one (Figure 1). A potential explanation is that samples containing less moisture retain more water upon rehydration to compensate for losses during storage. Water binding capacity is the ability of a material to retain free water in its structure. It depends on the physical and chemical properties of the specific material, including the structure of proteins and polymers. This is important for food, cosmetics, and other materials where humidity plays a key role. Water activity (aw) is a measure of the availability of free water, which can participate in various microbiological processes and chemical reactions in a given system. It is expressed as a ratio between the partial pressure of water in the system and the partial pressure of pure water at the same temperature [33].

The color brightness L* of dry egg products is directly related to the initial characteristics of the raw material and the applied heat treatment. The reported decrease in L* values may be a consequence of the Maillard reactions that took place during the drying process at high temperatures [32]. This type of reaction is also known as non-enzymatic browning of foods that have a high protein content. Color characteristic values can also be an indicator of changes in the product during drying. In the raw product, the L* value was around 66 (Table 2), but after drying, the value reached over 80 in three of the samples (Table 5). As a new method of processing this raw material, further research can be directed towards process optimization and reduction of pressing losses. It is also reasonable to monitor the chemical parameters during a longer period of storage and the factors affecting the AA profile in order to create a predictable product composition.

Changes in the lipid and protein fraction occurred during storage, but the values obtained for TBARS and PC were low. Some authors reported an increase in lipid oxidation and formation of TBARS in dried egg products with very low water activity (a_w < 0.300) [32]. In our case, we cannot conclude such a correlation. It is necessary to test these indicators for a longer period of storage since, after drying, the product is durable from a microbiological point of view, but the processes of changes in the lipid and protein fractions continue. These changes can lead to unacceptable changes in organoleptic indicators and the formation of a significant number of toxic substances [46].

Last but not least, the microbial status of the dried egg products from TA is stable during storage. This is most likely due to both the thermal treatment of the raw material and also the low moisture content and water activity. The absence of Salmonella spp. is a clear statement for the safety of the final products [47].

5. Conclusions

TA is a waste product with the potential to replace expensive protein raw materials in the feed of all categories of farm animals. European legislation allows its use for these purposes after appropriate treatment. The method based on acid–thermal coagulation reduces microbial contamination to an acceptable level. The process is characterized by low production losses, nutritional values are preserved during processing, and the resulting dry product is suitable for easy and long-term storage. It is necessary to carry out additional analyses during storage in order to determine the possible shelf life of the feed raw material. Coagulation of the product causes a phase transition of the product from liquid to solid. This allows the use of more versatile, simpler, and cheaper equipment for drying as compared to spray dryers. The method involves pre-dehydration of the material before drying, which can reduce energy consumption and the negative effect of heat on the product. Industrial tests should show the economic efficiency of this method and whether it will be used by egg processors. Larger scale tests should show the energy consumed for coagulation and drying of the product.