Analysis of the Fatty Acid Profile in Cream, Buttermilk Fractions, and Anhydrous Milk Fat: Influence of Physicochemical and Microbiological Parameters on the Fatty Acid Profile

Abstract

This study analyzes the impact of physicochemical parameters on the microbiological and fatty acid profiles of cream, buttermilk, and anhydrous milk fat. Using gas chromatography coupled with mass spectrometry (GC-MS), the fatty acids present in these dairy products were qualitatively determined, highlighting the differences associated with the processing stages. Specifically, the distribution of short-chain, medium-chain, and long-chain fatty acids, such as butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, oleic, and linoleic acids, were analyzed, assessing their nutritional implications. The microbiological composition of the samples was also analyzed using MALDI-ToF MS. The presence of lipolytic bacteria, such as Serratia monocytogenes, which can negatively impact the oxidative stability of fats, was detected. The results show that both environmental and processing conditions significantly affect the quantity and quality of fatty acids, thereby influencing the overall dietary value of dairy products. These findings offer insight into developing improved dairy product formulations that may lead to enhancement of their health benefits.

1. Introduction

Milk is one of the important products in the human diet. The fats that it contains are good energy sources and have a high bioavailability. There are also many milk products and derivatives available on the market, such as cream, sour cream, buttermilk, butter, and anhydrous milk fat (AMF) [1,2]. Each of these products is a subfraction of milk and is distinguished by the technological processes used to produce them. Cream is produced by mechanically separating the fat fraction from raw milk by centrifugation with heating. Skimmed milk, separated in the process, can be added to the separated product to obtain a product with the desired fat percentage. The product is then homogenized to prevent separation. It is also subjected to heat treatment through pasteurization or sterilization to maintain microbiological purity. In addition, dearomatization is also used to improve the properties and quality of the final product. In the final stage, the cream is cooled and aseptically packaged, in order to prevent possible contamination and preserve its freshness. It is equally important in the production of cream to achieve a product with a determined fat content. Therefore, milk, sour cream, and appropriate stabilizing additives are often combined. Such a mixture then undergoes homogenization and pasteurization [1,3,4]. In the case of cream, the product is cooled and inoculated with a starter culture of lactic acid bacteria (LAB) in the next step. These bacteria have an acidifying and flavoring effect. Bacteria used in the dairy industry include lactic bacteria of the species Lactococcus lactis subsp. lactis, L. lactis subsp. cremoris, and L. lactis subsp. lactis biovar. diacetylactis, as well as various species of the genus Leuconostoc. After adding the bacteria, the product is subjected to fermentation and then aseptically packaged [1,5]. Another subproduct of milk is anhydrous milk fat (AMF). It does not contain water or water-soluble components such as proteins and lactose in its composition. The process of obtaining, or clarification, involves the slow heating, melting, and cooking of butter, which leads to evaporation of the water component. The product is finally cleared of any foam and sediment that may have been formed [6]. Buttermilk is an aqueous phase extracted after churning cream into butter. Its composition is similar to that of skimmed milk, except that buttermilk contains a higher proportion of milk fat globule membrane components [7]. The quality and nutritional value of milk is determined by many chemical and physicochemical factors, such as fatty acid profile, milk efficiency, fat content, protein content, fat efficiency, protein efficiency, urea content, potential acidity, dry matter content, milk fat globule size, and density. These parameters are influenced by external factors such as the milking frequency and method, types of feed used, season, breed of cattle, health of cattle, stage of lactation, and lactation cycle [8,9,10]. The conducted study concentrated on analyzing the fatty acid profile of milk products. Milk fat mainly consists of tri-, di-, or monoesters of glycerol and fatty acids. Triacylglycerides alone account for as much as about 98.3% of the total fat content. In contrast, di- and monoesters together make up only about 0.3%. Also found in milk fat are phospholipids (0.8%), sterols (0.3%), and a small amount of free fatty acids (0.2%), as well as carotenoids and vitamins. To date, almost 500 different fatty acids have been identified in milk fat. However, only about 15 of them are found in amounts greater than 1%. Short-, medium-, and long-chain acids have been identified in milk fat [11]. We can find such short-chain acids as C4:0 (butyric acid), C6:0 (caproic acid), and C8:0 (caprylic acid). Medium-chain fatty acids that are found in milk include C10:0 (capric acid), C12:0 (lauric acid), and C14:0 (myristic acid). Long-chain acids found in milk fat include C16:0 (palmitic acid), C16:1 (oleopalmitic acid), C18:0 (stearic acid), C18:1 (oleic acid), C18:2 (linoleic acid), C18:3 (linolenic acid), and C20:4 (arachidic acid). Fatty acids found in milk fat can also be divided into saturated acids such as palmitic acid or stearic acid and mono- or polyunsaturated acids such as oleic acid, linoleic acid, and peanut acid, although the majority of fats found in milk are saturated [12,13,14]. Establishing the fatty acid content is crucial because of the health-promoting properties some of the fatty acids display. Clarified butter consists of about 63% saturated acids. Monounsaturated acids come in second at about 27%, followed by polyunsaturated acids at about 2.3%. About 25% of these lipids are short- and medium-chain fats, which are easily digestible by our bodies. Butyric acid itself shows anti-inflammatory, anticancer, and soothing properties [15]. In clarified butter, we can also find CLA, or conjugated linoleic acid diene, which is categorized as a health-promoting acid due to its anticancer properties and ability to lower body fat. Clarified butter, as a whole, has an anti-inflammatory effect and aids digestion, increases the absorption of vitamins, and strengthens immunity without raising cholesterol levels [6]. The previously mentioned external factors influence milk’s fatty acid content and physicochemical parameters. One study demonstrated that a cattle diet with a high grain feed content and low raw fiber, which is used to increase the protein content of milk, negatively affects the fat content of milk [16]. Other studies have reported that the breed of cattle has a significant effect on the parameters and quality of milk. The season also significantly affects these parameters, although the protein content and average fat globule size are the least affected. Milk obtained in winter was characterized by a higher concentration of basic components with a generally less favorable protein-to-fat ratio. Milk obtained in winter had a higher percentage of large fat globules (>10 μm) than in summer. Milk obtained in the summer period, when cows received pasture feed, had a 0.26% higher content of saturated fatty acids (SFAs). The content of unsaturated acids changed by 2.31%, including polyunsaturated acids alone by 0.51%. More favorable ratios of saturated acids to unsaturated ones (SFAs:UFAs) and monounsaturated and polyunsaturated acids to saturated acid content (MUFAs:SFAs and PUFAs:SFAs) were recorded during the summer. Most research papers emphasize that nutrition is one of the key factors determining the level and ratio of fatty acids in milk [15]. A study by Reklewska et al. [17] evaluating the content of eight bioactive fatty acids in milk from cows fed according to the total mixed ratio (TMR) system and grazed on pasture confirmed a significantly higher content of five acids (C18:1 t11, C18:2 c9 t11, C18:3, C20:4, and C20:5) in milk from cows grazed on pasture [8].

Thanks to advanced research, it was possible to precisely determine the fatty acid profile of various fractions of dairy products, including cream, buttermilk, and anhydrous milk fat samples. The results clearly showed that the quality of the raw material has a direct impact on the quality of the final product. The analysis showed that the distribution of fatty acids in cream samples and buttermilk fractions is strongly dependent on physicochemical parameters such as temperature, pH, and chemical composition, as well as on production and storage conditions. In addition, the presence of bacteria that exhibit proteolytic abilities can significantly affect the fatty acid profile by hydrolyzing fats and destabilizing emulsions. These bacteria, through their enzymatic activity, can lead to undesirable quality changes that directly affect the stability and nutritional value of dairy products. These findings underscore the critical importance of monitoring and controlling both raw material parameters and production processes to optimize the quality of final products. The application of rigorous quality control standards is essential to ensure the highest health and nutritional standards in the dairy industry. Systematic research and implementation of advanced analytical technologies can make a significant contribution to improving the quality and safety of dairy products while providing greater value to consumers.

2. Materials and Methods

2.1. Reagents and Chemicals

High-purity-grade chemicals were utilized in the study, including GCMS-grade water, acetonitrile, acetone, chloroform, sodium chloride, sulfuric acid (VI), hexane, methanol, potassium carbonate, trifluoroacetic acid, and formic acid (Sigma-Aldrich, Steinheim, Germany).

Organic matrices for matrix-assisted laser desorption/ionization of time of flight mass spectrometry (MALDI-ToF MS) such as α-cyano-4-hydroxycinnamic acid (HCCA) and bacterial test standard (BTS) were purchased from Bruker Daltonics (Bremen, Germany).

2.2. Sample Details

A variety of dairy product samples were used in our study. The analyses conducted included samples of cream, which is the raw material for the production of anhydrous milk fat, fractions of buttermilk, which is a by-product generated during production, and anhydrous milk fat, which is the final product. These samples were taken during three different process batches. Each sample was taken in a sterile manner from precisely marked locations, ensuring a high level of purity and minimizing the risk of contamination. Then, the samples were transported to the laboratory in special packaging that maintained a constant temperature of 4 °C, which ensured the stability and integrity of the samples until detailed laboratory analysis.

2.3. Microbiology Analysis

For microbiological analyses, culture media specified in ISO standards were used. The presence of microorganisms was determined according to the standards PN-ISO 15214:2002, PN-ISO 16649–2:2004, PKN/ISO/TS 22964:2008, and PN-EN ISO 7937:2005.

2.4. Extraction of Fatty Acids

The dairy product samples were obtained from a local dairy (Poland). Samples of cream, anhydrous milk fat (AMF), and buttermilk fractions were collected in sterilized glass containers. The procedure for methylating fatty acids (FAs) was based on the methods outlined by Christie [18] and Martínez et al. [19]. In order to identify the fatty acids contained in the samples, a derivatization process was carried out. To about 500 mg of sample, 2 mL of hexane was added and centrifuged at 3000× g for 15 min. After the specified time, the top hexane layer was collected, and the specified amount of hexane was added again. The process was repeated four times. The collected hexane layers were then evaporated under a stream of nitrogen, then 2 mL of 1% methanolic sulfuric acid solution was added to the obtained precipitant. The falcon tubes were then placed in a water bath and heated at 60 °C for 2 h. Then, the sample was cooled, and 2 mL of 6% aqueous sodium chloride solution was added to it and vortexed for 2 min. The sample was then extracted four times with 2 mL of hexane, collected, and the extracts combined. The final derivatization step involved neutralizing the combined hexane extracts with 3 mL of 6% aqueous potassium carbonate solution. After adding this solution, the top hexane layer was collected and evaporated under a stream of nitrogen. The resulting material was dissolved in 1 mL of methanol. The resulting solution was then diluted 1:100 in methanol and transferred to chromatography vials [20].

2.5. Characterization of Milk Fatty Acids Using GC-MS Analysis

Measurements were conducted using a 7820A gas chromatograph coupled with a 5977B GC/MSD mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a G4513A autosampler (Agilent Technologies, Palo Alto, CA, USA) and an HP-5MS column (30 m × 0.25 mm × 0.25 μm). The analysis was performed using a gradient temperature program. The injector temperature was 225 °C. The temperature program of the chromatographic oven was as follows: 50 °C (2 min), ramped at 13 °C/min to 150 °C (0 min), and then ramped at 5 °C/min to 250 °C (6 min). The helium flow rate through the column was 1.2 mL/min. Electron ionization (EI) at 70 eV was employed. The ion source temperature was 300 °C, and the transfer line temperature was 300 °C. MS spectra were recorded in the range of 35–550 m/z, and integrated peaks were identified using the NIST 17 library with a minimum match quality of 70%.

2.6. Identification of the Obtained Microorganisms by MALDI-ToF MS

The identification of isolated strains was conducted using MALDI-ToF MS (Microflex LT, Bruker Daltonics, Bremen, Germany) by directly applying the sample to the plate, according to the manufacturer’s procedure [21]. To achieve this, the isolated colony was spread thinly directly onto the sample position. Subsequently, 1 μL of 70% formic acid was applied to the sample; once dried, 1 μL of α-cyano-4-hydroxycinnamic acid matrix solution (10 mg/mL solution containing 50% acetonitrile, 47.5% water, and 2.5% trifluoroacetic acid) was applied. The collected spectra were analyzed using the Biotyper platform [22].

2.7. Statistical Analysis

Statistical analyses and the graphs were prepared with Microsoft Excel 365.

3. Results and Discussion

The study showed that the fatty acid profile of the cream samples and buttermilk fractions significantly depended on the physicochemical parameters [23,24,25].

In the first stage of the research conducted by our team, volatile organic compounds (VOCs) were determined in three cream samples divided into fractions (distinguished by numbered suffix, i.e., −1, −2, and −3), as presented in

Table 1. Reported volatile organic compounds in fractions from three cream samples, where 0—compound not detected; X—compound present in the sample (triplicate); LP—lipid number of fatty acid that takes the form C:D, where C is the number of carbon atoms in the fatty acid and D is the number of double bonds in the fatty acid; AMF—anhydrous milk fat. Fractions with no compound detected or only one from the list were omitted.

Retention Time (min)	Volatile Organic Compound	AMF-3	α-Serum Light Fraction-3	β-Serum Light Fraction-3	AMF-2	β-Serum Heavy Fraction-2	AMF-1	α-Serum Light Fraction-1
6.211	Butanoic acid (C4:0)	0	0	0	0	X	0	0
9.328	Hexanoic acid (C6:0)	0	0	0	X	0	0	0
12.475	Octanoic acid (C8:0)	X	0	X	X	0	0	0
16.363	Decanoic acid (C10:0)	X	X	X	X	0	X	X
20.692	Dodecanoic acid (C12:0)	X	0	X	X	0	X	X
24.91	(Z)-9-Tetradecenoic acid (C14:1)	X	0	X	X	0	0	0
25.01	Tetradecanoic acid (C14:0)	X	X	X	X	0	X	0
27.086	Pentadecanoic acid (C15:0)	X	0	X	X	0	0	0
28.85	(Z)-9-Hexadecenoic acid (C16:1)	X	0	X	X	0	0	0
29.109	Hexadecanoic acid (C16:0)	X	X	X	X	0	X	X
33.144	(E)-9-octadecenoic acid (C18:1)	X	0	X	X	0	0	0
33.58	Octadecanoic acid (C18:0)	X	0	X	X	0	0	0

.

Table 1 shows the reported volatile organic compounds in fractions from three investigated cream samples. A total number of thirteen VOCs were found, all of them belonging to fatty acids, including long-chain ones, such as octadecanoic acid (stearic acid), hexadecanoic acid (palmitic acid), and tetradecanoic acid (myristic acid). Ten VOCs were reported in the AMF-3 fraction, eleven VOCs in the AMF-2 fraction, and only four VOCs in the AMF-1 fraction. The cream fraction that had the closest qualitative composition to the AMF fraction—in this case, the AMF-3 sample—was the β-serum light fraction-3.

GC-MS analysis revealed the presence of various fatty acids, including palmitic, oleic, stearic, lauric, and myristic acids (Figure 1). The composition of these acids differed be-tween samples, which may be the result of differences in processing and storage conditions. Cream samples 1 and 2 showed significant differences in oleic acid and myristic acid content (Table 1). Such differences may be due to different production and storage conditions, which affect the stability and degradation of individual fatty acids. We also noted (Figure 1) that the cream 3 sample contained almost exclusively palmitic acid, as well as significant amounts of capric and myristic acids. It indicates a possible specific production method or selective removal of other acids. In addition, the β-serum light-3 sample was the richest in both quantity and quality compared to the other samples (Table 1), as it contained significant amounts of fatty acids also present in the AMF-3 sample, suggesting that it may be a valuable source of these compounds.

Figure 1 confirms the above statement that the richest (i.e., the most qualitatively diverse) and closest in composition cream fraction was the β-serum light fraction-3. Subsequently, these were α-serum light fraction-3 and α-serum light fraction-1, in relation to the appropriate AMF samples. Also, the most common VOC in all samples was hexadecanoic acid (palmitic acid), followed by (E)-9-octadecenoic acid (elaidic acid; example of omega-9 fatty acid), tetradecanoic acid (myristic acid), and octadecanoic acid (stearic acid).

The results of Khan et al. [26] showed that the storage method of dairy products has a significant impact on the antioxidant properties, fatty acid profile, and lipid oxidation. Temperature, light, and storage time have a significant effect on maintaining the quality of fats in dairy products, as their inadequate storage can lead to fatty acid oxidation, which was noted in the case of our study. These factors lead to significant changes in the protein and fat composition of milk. Understanding the complexity of these changes is key to understanding their impact on the functionality, sensory characteristics, and nutritional value of milk, which are directly relevant to the quality of this essential product [20,27]. Comparison of the content of individual fatty acids in different samples revealed that cream samples 1 and 2 had similar profiles but with differences in the proportions of individual acids (Tables S3 and S4). On the other hand, cream sample 3 was significantly different from the others, which may be the result of a specific production or storage method (Table S5). In addition, microbiological analysis confirmed our assumptions about the stability of the fat (

Table 2. List of identified microorganisms in buttermilk fractions and anhydrous milk fat with the MALDI-ToF MS technique.

Nr.	Name Sample	Microorganisms	MSP Score *
1	Part 1
	α–serum light fraction	Pseudomonas fluorescens Bacillus cereus Serratia marcescens Escherichia coli Micrococcus luteus Staphylococcus aureus	2.21 2.18 2.32 2.08 2.36 2.19
	α–serum heavy fraction	Micrococcus luteus	2.03
	β-serum light fraction	Pseudomonas fluorescens Lactococcus raffinolactis Serratia marcescens Micrococcus luteus Escherichia coli	2.14 2.23 2.45 2.07 2.06
	β-serum heavy fraction	Micrococcus luteus Staphylococcus aureus	2.29 2.32
	AMF **	no identified microorganisms
Part 2
2	α–serum light fraction	Micrococcus luteus Serratia marcescens	2.20 2.37
	α–serum heavy fraction	Micrococcus luteus Staphylococcus aureus Lactococcus raffinolactis Escherichia coli	2.12 2.21 2.35 2.41
	β-serum light fraction	Serratia marcescens Micrococcus luteus	2.40 2.27
	β-serum heavy fraction	Pseudomonas fluorescens Staphylococcus aureus	2.26 2.19
	AMF **	no identified microorganisms
Part 3
3	α–serum light fraction	Micrococcus luteus Escherichia coli	2.22 2.36
	α–serum heavy fraction	Pseudomonas fluorescens Lactococcus raffinolactis	2.20 2.16
	β-serum light fraction	Pseudomonas fluorescens Lactococcus raffinolactis	2.36 2.14
	β-serum heavy fraction	Lactococcus raffinolactis Micrococcus luteus	2.02 2.26
	AMF **	no identified microorganisms

* Log score >2.00—secure genus identification, probable species identification. ** Anhydrous milk fat.

). Physicochemical properties (Table S1) may induce changes occurring at late stages of acidification, including fatty acid profiling, pH parameter, color, or production of bioactive compounds [28].

Figure 2 shows the presence of fatty acids among all the tested samples. Three compounds were the most predominant, namely butanoic acid (butyric acid), decanoic acid (capric acid), and hexadecanoic acid (palmitic acid). Hexanoic acid (caproic acid) was a fatty acid found only once in the sample of AMF-2.

A study by Pasvolsky et al. [29] identified free fatty acids (FFAs), including butyric acid, as a component of milk that can induce biofilm formation by microorganisms present in milk. They observed that Bacillus cells were in close proximity to milk fat globules. However, not only Bacillus sp. bacteria can form biofilm but also Escherichia coli and Staphylococcus aureus, as well as lactic acid bacteria (LAB) [30,31,32,33]. Consequently, the accumulation of the aforementioned FFAs adduces to the hydrolysis of milk fat, a process referred to as lipolysis [34,35]. This process can occur through enzymatic activity or spontaneously during milk production [36]. Many bacteria of the genus Bacillus produce lipases capable of lipolyzing milk fat, some of which retain activity even after pasteurization [37,38]. Therefore, because Bacillus bacteria actively lipolyze milk fat, when they are present in milk, they deplete its milk fat level. A similar relationship is observed for our microbiological analyses, as they revealed the presence of various bacterial species, including E. coli, Serratia liquefaciens, L. lactis, and Micrococcus luteus (Table 2). These bacteria may have proteolytic and lipolytic abilities, which can affect the fatty acid profile of the samples tested. In a study by Parkash et al. [39], M. luteus bacteria showed slow proteolytic activity compared to other bacteria, e.g., B. cereus, P. aeruginosa, and S. marcescens. In contrast, isolates of B. cereus, M. luteus, P. aeruginosa, S. marcescens, and S. aureus showed lipolytic activity, except for E. coli. A study by the Baur et al. team [40] showed that numerous microbial isolates from raw milk, including bacteria but also yeast, produce extracellular enzymes that can cause spoilage problems in the dairy industry. Bacterial lipases are widely used in the food industry, especially dairy [41]. Numerous bacterial species can produce these enzymes, including B. licheniformis, P. fluorescens, and Acinetobacter sp. AU07 [42,43,44]. Among psychrotrophic bacteria that produce lipolytic enzymes, Pseudomonas is the predominant genus among G(−) bacteria, while Bacillus is the predominant G(+) genus [45].

Other types of psychrotrophic bacteria that can also produce lipolytic enzymes are Serratia, Hafnia, Microbacterium, and Enterobacter [46]. Bacteria of the genus Serratia, present in cream fractions, can hydrolyze triacylglycerols to FFAs, leading to a change in their profile (Table 2), as confirmed in a study by Salgado et al. [47]. These enzymes can hydrolyze triacylglycerols present in foods, generating FFAs responsible for unpleasant perceived flavors, e.g., rancid, buttery, but also pungent, bitter, soapy, or even astringent, flavor [35,45]. A team of researchers, Hantsis-Zacharov and Halpern [48], demonstrated that many lipolytic isolates belong to the genus Pseudomonas. In their study, more than 70% of Pseudomonas isolates showed lipolytic activity. The secretion of extracellular lipases has been described for several Pseudomonas species, such as P. pseudoalcaligenes and P. fluorescens [49,50]. Thus, the presence, as well as the proteolytic capacity, of some of the bacteria we identified could also affect the stability of fat emulsions, which post-mediately affected the composition of FFAs, as confirmed by GC-MS analyses (Table 1).

Through advanced research, it was possible to precisely determine the fatty acid profile of cream, buttermilk fractions, and anhydrous milk fat samples. The results unequivocally demonstrated that the quality of the raw material directly impacts the quality of the final product. It was found that the distribution of fatty acids in cream samples, buttermilk fractions, and AMF is strongly dependent on the physicochemical parameters, as well as production and storage conditions. Furthermore, the presence of bacteria exhibiting lipolytic and proteolytic abilities can significantly influence the fatty acid profile by hydrolyzing fats and destabilizing emulsions. These bacteria, through their enzymatic activities, can lead to undesirable qualitative changes that directly affect the stability and nutritional value of dairy products. These findings underline the crucial importance of monitoring and controlling both raw material parameters and production processes to optimize the quality of the final products. Such an approach is essential for ensuring the highest health and nutritional standards in the dairy industry.