Effects of Microfiltration and Storage Time on Cholesterol, Cis-9, Trans-11 and Trans-10, Cis-12 Conjugated Linoleic Acid Levels, and Fatty Acid Compositions in Pasteurized Milk

Abstract

The effects of microfiltration and storage time on the contents of cholesterols, conjugated linoleic acids, particle size distributions, and fatty acid profiles in pasteurized milk were investigated over 7 days. After microfiltration, the cholesterol (except for day 7) and trans-10, cis-12 conjugated linoleic acids did not change in microfiltered and pasteurized milk and in pasteurized milk. Compared with pasteurized milk, the cis-9, trans-11 conjugated linoleic acids, and ω-6/ω-3 ratio decreased by 43 and 12%, while d₃₂, d₄₃, C18:3n-3, and C20:5n-3 increased by 52–57, 70, 3.2–5.8, and 4.8–2.6% in microfiltered and pasteurized milk, respectively. The contents of cis-9, trans-11 conjugated linoleic acids, and cholesterols were higher on day 7 than on day 0, while the ω-6/ω-3 ratio and C22:6n-3 showed the opposite tendencies. Storage time did not affect d₃₂ (except for microfiltered and pasteurized milk) and d₄₃. These highlighted that microfiltration has the potential to retain ω-3 fatty acids, decreased the ω-6/ω-3 ratio and maintain the stability of microfiltered and pasteurized milk shelf life.

Keywords:

• Microfiltration
• Fat globule size
• Conjugated linoleic acids
• Cholesterol
• Shelf life
• Milk

INTRODUCTION

Microfiltration (MF) is a new treatment process applied to dairy industry, which is primarily used for bacterial removal, whey defatting, casein concentration, and fat selective separation.^[1] Many researchers have studied how MF affects the microorganisms, proximate compositions, organoleptic properties, and shelf life of dairy products.^[2–4] Only few researchers have used MF to separate different fractions of the initial milk fat globule population, obtaining fatty acid fractions of the native milk fat globules of different sizes.^[5–9] However, no reports have demonstrated the effect of MF on the contents of conjugated linoleic acids (CLAs) and cholesterols and the size distribution of fat globules in pasteurized milk (PM) during chilled storage.

Fat is one of the most abundant components of milk and dairy products, and its content and composition are more influenced by nutrition, environmental conditions, and processing methods.^[10–12] CLAs are fatty acids that are a group of conjugated or non-methylene interrupted dienoic 18:2s with double bonds which are located in the positions from 6 and 8 to 12 and 14.^[10,13] Among eight CLA isomers, the biological activities of CLAs have been attributed to two main isomers, cis-9, trans-11 and trans-10, cis-12 either alone or synergistically.^[7] CLAs can reduce risk of carcinogenesis and atherosclerosis, depress cholesterols, modulate immune system functions in humans, and possess anti-obesity and anti-tumor activities.^{[10–11,14,15]} Consequently, these CLA-rich products are healthy foods for consumers. The richest sources of CLAs in our diet are milk and dairy products, and cow milk fat is the largest natural source of the cis-9, trans-11 isomer, which represents 80–90 g/100 g of the total CLAs.^[11,13] Previous studies indicated that the effects of processing on CLAs in dairy products were associated with different processing technologies and treatment conditions, for example, heating can change the CLA isomer distribution in dairy products and microwaving results in 53% loss of CLAs.^[10] However, not much work has been done on the effects of MF and storage time on CLAs in PM.

The fat content and composition in the diet are also key factors affecting serum cholesterol content.^[13,16–18] For instance, saturated fatty acids (SFAs) accounting for 65% of milk fat may raise total and low-density lipoprotein (LDL) cholesterols, increasing the risk of cardiovascular disease.^[11,13,19] Low levels of trans isomers of fatty acids (FAs) in dairy products have also been implicated in raising human serum cholesterol levels. Cholesterol is the major sterol, 95% of total, and is an important compound in milk and acts as a critical component of cell membranes, the precursor of all steroid hormones, and one of the precursors of vitamin D. On the other hand, consumption of more cholesterol has been related to the incidence and severity of cardiovascular incidents.^[18] Moreover, the cholesterol in milk was found—together with phospholipids—in the hydrophilic surface layer of fat globules.^[20] The size (diameter) of milk fat globules has a crucial impact on the digestibility of milk fat and its nutritional value.^[16,21] Therefore, it is important to determine the cholesterol content and size distribution of fat globules as well as their relationships in milk and dairy products, because milk products have been designated as the major contributors to dietary cholesterol. Processing technologies and storage conditions can effective decrease the SFAs content and multiply concentrations of bioactive lipids (e.g., CLAs and/or omega-3 FAs) in milk, however, the effects of MF and storage on the cholesterol content, fat globule size distribution, and their relationships in dairy products are not very clear.^[9]

The quality and functionality of milk products are closely related to the processing methods used. Therefore, to ensure that MPM and PM milks supply consumers with all nutrients in their most available form, comply with their requirements and maintain their organoleptic properties, it is necessary to comprehensively monitor and compare the changes in nutritional components present in milk during different processing and storage conditions. Therefore, the purpose of this study was to investigate whether the MF and storage time affect the content of cis-9, trans-11 and trans-10, cis-12 CLAs and cholesterols, the profile of FAs, and the size distribution of fat globules in the microfiltered and pasturized milk (MPM) after processing, as well as during 7 days of storage at 4°C, and to determine the relationships among all indexes tested in this study. Conventionally PM was used as a comparator.

MATERIALS AND METHODS

Raw Milk and Milk Processing

Fresh raw milk, collected from the Jinshan dairy farm (Shanghai, China), was centrifuged at 4000 × g for 30 min to remove the fat and leucocytes .The skimmed milk (less than 0.1% fat) was microfiltered through ceramic membranes (Tami Industries, France; nominal pore size: 1.4 μm; total membrane surface area: 13.3 m²; flow rate; 5t·h^–1; inlet pressure: 2.5 bar) at 50°C. The cream stream (35% fat) was heated at 120°C for 4 s and then mixed with the permeate of MF to form the fat-adjusted whole milk (3.4% fat). The mixture was then homogenized (150/50 bar) at 65°C and pasteurized at 72°C for 15 s, cooled to 4°C and then bottled to give a stable product for analysis.^[22] The shelf life of this MPMis 7 days, which was determined by the Bright Dairy & Food Co., Ltd (China) accordingto the Chinese National Standards for dairy products. Analysis and testing were conducted at 0, 4, and 7 d.

Particle Size Measurements

The fat globule size distribution of the milk was estimated by laser light scattering using a Malvern Mastersizer 3000 (Malvern Instruments, Malvern, UK). The samples were diluted in distilled water until an appropriate obscuration was obtained in the measurement cell. The stirred mixture was then continuously recycled through the sample cell of the Malvern with a laser wavelength of 633 nm. An optical model based on the Mie theory of light scattering by spherical particles was applied using the following conditions: real refractive index of 1.520; refractive index of fluid (water) of 1.330; and a pump speed of 21%. The fat globule size distribution of the milk was characterized by standard parameters: the volume-surface average diameter d₃₂ (; where is the volume of globules in a size class of average diameter) and the volumic average diameter d₄₃ ().^[7]

Total Fat Analysis

The contents of total fat were determined according to Association of Official Analytical Chemists (AOAC) method.^[23]

Determination of Cholesterol

Total cholesterol contents were determined by liquid chromatography following the Chinese standard method GB/T 22220-2008.^[24] An Alliance 2965 system (Waters Corp., Medford, MA, USA) liquid chromatograph system equipped with an ultra violet (UV) detector at 206 nm was used. The chromatographic separation was carried out on a reversed-phase C18 column (5 μm, 3.2 × 250 mm, Waters Corp., Medford, MA, USA.) and the mobile phase was methanol with a flow rate at 1.0 mL/min. Column temperature was set at 35°C and the sample injection volume was 10 μL.

Lipid Extraction and Preparation of Fatty Acid Methyl Esters

Lipid extraction and preparation of fatty acid methyl esters were performed according to the previous method with modifications.^[7] Briefly, freeze-dried milk samples (1.5 g) were mixed with 10 mL hexane: diethyl ether (1:1, v/v) mixture, 2 mL of saturated NaCl solution and 1 of ethanol solution. After homogenization at 2350 × g for 10 min, the upper layer was recovered, dehydrated with anhydrous sodium sulphate (Na₂SO₄), concentrated using a rotary evaporator at 35°C to a final volume of approximately 5 mL, flushed with nitrogen until dry, and stored at –75°C for further use. To prepare fatty acid methyl esters, approximately 100 mg of the total milk fat was dissolved in 6 mL of hexane. After the addition of 18 μL of sodium methoxide, the mixture was vortexed and let in contact for 5 min. Anhydrous sodium sulfate was added before centrifugation at 1000 × g for 10 min at 20°C. The upper layer was recovered and 1 mL was diluted 10 times in hexane before injection into the gas chromatograph (GC).

GC Analysis

The instrumentation used for the determination of CLAs was an Agilent 6890 GC with fused-silica capillary column with 100% bis (3-cyanopropyl) polysiloxane (~100 m length, 0.25 mm i.d., 0.20 μm film thickness) and a flame ionization detector. Operational conditions were as follows: the injection volume was 1 mL; the injector and detector temperatures were 200 and 250°C, respectively; nitrogen was used as the carrier gas and the flow rate was 1 mL/min. The oven temperature was programmed as follows: 40°C for 5 min then the temperature was increased to 220°C at a rate of 20°C min^–1 and left at 220°C for 40 min. The sample splitting ratio was 30:1. The CLA peaks were identified by comparison with the retention times of the reference standard (CLA methyl ester, Sigma Chemical Co., St. Louis). The fatty acid identification was performed on the same sample prepared for the analysis of CLA using the same analysis conditions. Peak identification was possible with the aid of an external standard (FIM-FAME-7 mixture: Matreya, Inc., Pleasant Gap, PA, USA; 30 mg/mL; 37 components).

Data Analysis

Statistical analysis of variance (ANOVA) was performed using SAS 8.0 statistical data analytical software (SAS Inst., Inc., Cary, NC, USA). Significant differences between means were determined by a least significant difference (LSD) test procedure at p < 0.05.

RESULTS AND DISCUSSION

Effects of MF and Storage Time on Particle Size Distribution

The average diameter of milk fat globules has a remarkable contribution to the technological suitability and nutritional value of milk.^[16] Particle size parameters for the different milks are shown in Table 1. A small peak at 0.2 μm that corresponded to casein micelles and a main peak at 3.7 μm that corresponded to fat globules characterized the particle size distribution curve of raw milk.^[25] Generally speaking, raw milk has only one peak after homogenization, and the mode depends on the pressure of the homogenization. The particle size distribution curve of PM was characterized by one peak at 0.719 μm, whereas MPM showed the presence of a second small at small peak at higher diameters reflecting the formation of large casein particles or fat aggregates, whose peaks were at 0.719 and 2.580 μm, respectively. These clusters can be formed through shared casein adsorbed onto the fat globules membrane or by coalescence of fat globules.^[6] The d₃₂ parameter is sensitive to small particles. The smaller the value of the d₃₂ parameter, the smaller particles. The d₄₃ parameter is very sensitive to the presence of large particles. A small amount of large particles could cause a large change in the d₄₃ parameter. MPM showed an increase in d₄₃, which reflects the formation of the previously mentioned aggregates. Fat globule size parameters of MPM and PM after 7 days of cold storage showed no significant changes, indicating that the treated milk was stable during the shelf life.

TABLE 1 Fat globule size distribution of milk during shelf life

	d32 (μm)			d43 (μm)
	0 d	4 d	7 d	0 d	4 d	7 d
PM	0.50 ± 0.01^Ab	0.48 ± 0.01^Ab	0.48 ± 0.00^Ab	0.70 ± 0.00^Ab	0.70 ± 0.00^Ab	0.69 ± 0.01^Ab
MPM	1.15 ± 0.04^Aa	1.08 ± 0.03^Ba	1.08 ± 0.04^Ba	2.32 ± 0.08^Aa	2.24 ± 0.09^Aa	2.29 ± 0.12^Aa

PM: pasteurized milk; MPM: microfiltered and pasteurized milk; d₃₂: volume-surface average diameter; d₄₃: volumic average diameter; Values were expressed as average ± standard error (SE); Means in same row with same online capital letters were not significantly different (p < 0.05); Means in same column with same online lowercase letters were not significantly different (p < 0.05).

Effects of MF and Storage Time on Total Fat and Cholesterol Contents

Table 2 shows the fat and cholesterol contents of MPM and PM analyzed at the various storage times. The fat contents were not significantly different between PM and MPM (p > 0.05). No statistically significant change were also found in all milk samples during storage (p > 0.05), indicating that there was no significant lipolysis during the storage period. These results agree with a previous report on sheep milk yogurts.^[9]

TABLE 2 The contents of total fat, cholesterol, cis-9, trans-11, and trans-10 and cis-12 conjugated linoleic acids (CLAs) of milk during shelf life

	Total fat (g/100 g milk)			Cholesterol (mg/100 g milk)			cis-9, trans-11 CLA (mg/100 g milk)			trans-10, cis-12 CLA (mg/100 g milk)
	0 d	4 d	7 d	0 d	4 d	7 d	0 d	4 d	7 d	0 d	4 d	7 d
PM	3.41 ± 0.01^Aa	3.39 ± 0.03^Aa	3.42 ± 0.02^Aa	12.17 ± 0.15^aB	13.37 ± 0.21^aA	13.57 ± 0.15^aA	16.77 ± 0.59^Ba	17.38 ± 0.27^Ba	18.33 ± 0.11^Aa	0.51 ± 0.02^Ca	0.54 ± 0.00^Ba	0.58 ± 0.01^Aa
MPM	3.42 ± 0.03^Aa	3.42 ± 0.04^Aa	3.32 ± 0.05^Aa	12.30 ± 0.52^aB	13.63 ± 0.21^aA	12.77 ± 0.23^bB	9.56 ± 0.39^Bb	10.08 ± 0.49^ABb	10.52 ± 0.08^Ab	0.51 ± 0.01^Ca	0.53 ± 0.01^Ba	0.56 ± 0.01^Aa

PM: pasteurized milk; MPM: microfiltered and pasteurized milk; Values were expressed as average ± standard error; Means in same row with same online capital letters were not significantly different (p < 0.05); Means in same column with same online lowercase letters were not significantly different (p < 0.05).

It can also be seen that there was no significant difference (p > 0.05) in the cholesterol content between MPM and PM at the same storage time, with the only exception of the cholesterol content which was lower in MPM than in PM on day 7. Levels of cholesterols in PM were lower on day 0 than on other days, and no significant differences (p > 0.05) were found between on day 4 and on day 7. For MPM, the cholesterol contents were the highest on day 4, and there were no significant differences in cholesterol levels between on day 0 and day 7. The results obtained showed that the MF had little influence on the cholesterol content in milk. Ali and Abdel-Razig found that the cholesterol level of Mozzarella cheese increased significantly (p <0.05) with storage time.^[20] In present study, no explanation was provided on why the cholesterol contents increased with storage time, which should be investigated in our future work. However, Baggio and Bragagnolo mentioned that the storage time did not alter the cholesterol contents of the processed meat products throughout the 90-day storage period.^[26] These different conclusions could be associated with the physiological fluctuations of cholesterol content of milk, food matrix, fatty acid composition, processes, and treatment applied. In this study, the cholestrol content was positively but non-significantly correlated with total fat content, as well as negatively but non-significantly correlated with d₃₂ (r = –0.148) and d₄₃ (r = –0.116; p < 0.01). The relationship between cholesterol and trans-10, cis-12 CLA was highly significant (r = 0.61, p < 0.01). Šterna and Jemeljanovs found that the concentration of cholesterol increases along with the increasing fat content.^[27] Ali and Abdel-Razig also stated that the cholesterol/fat ratio varies tremendously according to globule size, being highest in the smallest globules.^[20] Moreover, Barlowska et al. claimed that contents of fat and cholesterol were negatively correlated (r = –0.09*).^[16]

Effects of MF and Storage Time on Cis-9, Trans-11 and Trans-10, Cis-12 CLAs

The cis-9, trans-11 and trans-10, cis-12 CLAs in MPM and PM samples are given in Table 2. The cis-9, trans-11 CLA contents in PM and MPM samples ranged from 16.77 to 18.33 mg/100 g milk (i.e., 4.91 to 5.35 mg/g fat) and 9.56 to 10.52 mg/100 g milk (i.e., 2.80 to 3.17 mg/g fat), respectively. The trans-10, cis-12 CLAs occurred only in low amounts and changed between 0.51 and 0.58 mg/100 g milk (i.e., 0.15 to 0.17 mg/g fat). Similarily, Seçkin et al. stated that that the cis-9, trans-11 CLAs concentration in processed cheese, Kaymak and cream samples varied from 1.50 to 7.946 mg/g fat.^[13] Michalski et al. reported that the amount of the trans-10, cis-12 is less than 0.01% of total FAs (i.e., ˂0.6% of total CLAs) in milk.^[7] The amount of cis-9, trans-11 CLAs was higher in PM than in MPM, but that of trans-10, cis-12 CLAs did not markedly alter between in MPM and in PM at the same storage time (p > 0.05). This indicated that MF decreased the cis-9, trans-11 CLAs. This phenomenon could attribute to (1) greater volumic average diameter (d₄₃) and volume-surface average diameter (d₃₂) of globules in MPM than in PM (Table 1), (2) lower cis-9, trans-11 isomer in the large fat globules,^[13] and (3) selective separation of milk fat in small globules and in large globules (fat globule relative diameter is 15–0.2 mm) through a ceramic membrane with an average pore size of 1.4 μm.^[1] The content of cis-9, trans-11 CLAs non-significantly increased from day 0 to 4 of storage, and noticeably increased thereafter. The trans-10, cis-12 CLAs increased significantly with storage time in PM and MPM. Gursoy et al. also found that the cis-9, trans-11 CLAs contents increased during 90 days of ripening in pickle white cheeses produced from whole milk with four different probiotic cultures (Enterococcus faecium, Lactobacillus paracasei, Bifidobacterium longum, and Lactobacillus acidophilus).^[28] However, it has been suggested that aged cheeses have lower amount of CLAs than those with a shorter ripening period.^[15] Moreover, no changes in CLAs were observed after storing dairy products at low temperatures or following some processing methods.^[29] This discrepancy could be related to the geographical origin, seasonal variations, and the breeding methods of dairy cattle, initial CLA content of raw milk, temperature, type of starter cultures, production, and ripening process.^[9,10,13,28] We can offer no satisfactory explanation for these alterations in this study. We speculate that an increase in CLAs might be due to the lipolysis of free linoleic acid and the conversion of vaccenic acid to CLAs.^[15,28,29] Significantly positive correlation between the content of the cis-9, trans-11 CLAs and that of total fat (r = 0.974, p < 0.01) and negative correlation for d₃₂ (r = –0.985) and d₄₃ (r = –0.983; p < 0.01) were observed in the present study.

Effects of MF and Storage Time on FAs Composition

Sixteen SFAs and 16 unsaturated fatty acids (UFAs) identified in the milk were segregated into short- (C4 to C7), medium- (C8 to C14), and long-chain (C15 to C24) FAs, according to their carbon-chain lengths (Table 3).At the same storage days, the contents of C13:0, C16:0, C16:1, C18:3n-3, and C20:5n-3 were significant greater in MPM than in PM, while the concentrations of the other FAs were lower in PM than in MPM, which was caused by selective fat removal through the use of ceramic MF membranes.^[1] Among all SFAs, C16:0 was the most abundant, and followed by C18:0 and 14:0 in milk. These results are similar to previous reports.^[13] C16:0 is one of the major SFAs; it raises serum cholesterol while C18:0 does not.^[30]

TABLE 3 Fatty acid composition of milk during shelf life (mg/100 g milk)

	PM			MPM
Fatty acids	0 d	4 d	7 d	0 d	4 d	7 d
C4:0	66.85 ± 0.77^a	66.17 ± 0.46^ab	61.85 ± 0.80^d	61.54 ± 1.18^d	65.07 ± 0.56^b	63.60 ± 0.13^c
C6:0	50.10 ± 0.30^a	49.16 ± 0.08^b	48.64 ± 0.20^bc	47.29 ± 0.61^d	48.44 ± 0.16^c	47.79 ± 0.49^d
C8:0	32.35 ± 0.04^a	31.75 ± 0.13^b	31.93 ± 0.20^b	30.36 ± 0.24^d	30.83 ± 0.05^c	30.40 ± 0.36^d
C10:0	70.90 ± 0.05^a	69.77 ± 0.38^b	70.28 ± 0.52^ab	65.80 ± 0.31^d	66.69 ± 0.03^c	65.72 ± 0.63^d
C11:0	7.45 ± 0.01^a	7.33 ± 0.01^b	7.36 ± 0.13^ab	7.04 ± 0.01^d	7.18 ± 0.01^c	6.98 ± 0.03^d
C12:0	77.28 ± 0.01^a	76.05 ± 0.42^b	76.77 ± 0.80^ab	71.06 ± 0.06^c	71.48 ± 0.18^c	70.92 ± 0.54^c
C13:0	4.98 ± 0.02^ab	4.85 ± 0.10^c	4.85 ± 0.02^c	5.09 ± 0.06^a	5.02 ± 0.05^ab	4.93 ± 0.08^bc
C14:0	246.33 ± 0.39^a	241.70 ± 1.52^b	243.33 ± 1.00^b	230.39 ± 0.10^c	231.09 ± 0.80^c	229.89 ± 1.83^c
C14:1	18.18 ± 0.06^a	17.87 ± 0.11^b	17.98 ± 0.07^b	17.10 ± 0.01^c	17.13 ± 0.04^c	17.04 ± 0.09^c
C15:0	24.04 ± 0.07^a	23.57 ± 0.15^b	23.73 ± 0.07^b	23.21 ± 0.01^c	23.33 ± 0.08^c	23.29 ± 0.21^c
C16:0	739.25 ± 3.03^b	722.56 ± 4.47^c	728.37 ± 1.35^c	755.77 ± 0.18^a	757.72 ± 2.96^a	755.43 ± 7.41^a
C16:1	40.62 ± 0.29^b	40.24 ± 0.25^b	40.43 ± 0.10^b	41.23 ± 0.01^a	41.36 ± 0.17^a	41.16 ± 0.43^a
C17:0	15.53 ± 0.04^a	15.21 ± 0.11^b	15.31 ± 0.01^b	14.81 ± 0.01^c	14.86 ± 0.04^c	14.78 ± 0.15^c
C18:0	313.09 ± 1.87^a	305.15 ± 2.20^c	308.62 ± 0.24^b	300.75 ± 0.07^d	301.58 ± 0.84^d	300.54 ± 2.32^d
18:1, trans	53.36 ± 0.37^b	54.15 ± 0.29^a	51.39 ± 0.53^c	48.07 ± 0.12^e	49.29 ± 0.01^d	48.49 ± 0.04^e
C18:1n-9	585.14 ± 3.32^a	567.82 ± 4.41c	576.27 ± 0.72^b	573.20 ± 0.39^bc	573.45 ± 2.32^bc	571.56 ± 5.32^bc
18:2, trans	14.16 ± 0.01^c	14.48 ± 0.10^b	14.66 ± 0.08a	13.79 ± 0.06^d	13.83 ± 0.01^d	13.77 ± 0.04d
C18:2n-6, cis	86.33 ± 0.50^a	83.14 ± 0.72^c	84.57 ± 0.65^b	77.72 ± 0.01^d	77.60 ± 0.35^d	77.54 ± 0.74^d
C20:0	3.71 ± 0.02^a	3.61 ± 0.02^c	3.65 ± 0.01^b	3.49 ± 0.01^e	3.53 ± 0.01^d	3.51 ± 0.03^de
C20:1	1.27 ± 0.01^a	1.21 ± 0.01^b	1.18 ± 0.03^cd	1.21 ± 0.02^bc	1.16 ± 0.01^d	1.17 ± 0.02^d
C18:3n-3	9.29 ± 0.07^b	9.06 ± 0.07^c	9.20 ± 0.04^b	9.60 ± 0.02^a	9.62 ± 0.04^a	9.61 ± 0.09^a
C20:2	0.75 ± 0.01^a	0.74 ± 0.01^a	0.74 ± 0.01^a	0.65 ± 0.02^b	0.64 ± 0.01^b	0.65 ± 0.01^b
C22:0	1.76 ± 0.01^a	1.69 ± 0.02^b	1.70 ± 0.01^b	1.57 ± 0.01^c	1.57 ± 0.01^c	1.55 ± 0.03^c
C20:3n-6	5.82 ± 0.03^a	5.70 ± 0.04^b	5.75 ± 0.02^b	5.39 ± 0.03^c	5.40 ± 0.04^c	5.38 ± 0.05^c
C22:1n-9	0.57 ± 0.01^a	0.57 ± 0.01^a	0.56 ± 0.01^a	0.23 ± 0.01^c	0.25 ± 0.01^b	0.25 ± 0.01^b
C20:3n-3	0.17 ± 0.01^b	0.18 ± 0.01^a	0.18 ± 0.01^ab	0.11 ± 0.01^d	0.13 ± 0.01^c	0.13 ± 0.01^c
C20:4n-6	5.36 ± 0.03^a	5.20 ± 0.06^c	5.28 ± 0.01^b	4.87 ± 0.02^d	4.84 ± 0.02^de	4.80 ± 0.03^e
C23:0	1.40 ± 0.01^a	1.37 ± 0.02^b	1.39 ± 0.01^ab	1.20 ± 0.01^e	1.27 ± 0.01^c	1.23 ± 0.02^d
C24:0	1.42 ± 0.02^a	1.39 ± 0.02^b	1.41 ± 0.01^ab	1.26 ± 0.01^c	1.27 ± 0.01^c	1.27 ± 0.02^c
C20:5n-3	0.79 ± 0.02^c	0.76 ± 0.01^c	0.79 ± 0.02^c	0.85 ± 0.02^ab	0.87 ± 0.03^a	0.83 ± 0.02^b
C24:1	0.28 ± 0.01^a	0.28 ± 0.01^a	0.28 ± 0.01^a	0.23 ± 0.01^b	0.23 ± 0.01^b	0.21 ± 0.01^c
C22:6n-3	0.14 ± 0.01^b	0.11 ± 0.01^e	0.12 ± 0.01^d	0.15 ± 0.01^a	0.12 ± 0.01^d	0.13 ± 0.01^c
SFA	1656.45 ± 4.29^a	1621.33 ± 10.09^b	1629.15 ± 5.38^b	1620.64 ± 2.27^b	1630.94 ± 4.05^b	1621.95 ± 13.85^b
MUFA	699.41 ± 4.03^a	682.14 ± 5.05^bc	688.09 ± 0.40^b	681.27 ± 0.49^bc	682.87 ± 2.54^bc	679.88 ± 5.80^c
PUFA	122.81 ± 0.59^a	119.38 ± 0.99^c	121.29 ± 0.61^b	113.13 ± 0.14^d	113.07 ± 0.50^d	112.84 ± 0.97^d
Σn-3	10.39 ± 0.05^b	10.11 ± 0.07^c	10.29 ± 0.06^b	10.71 ± 0.04^a	10.75 ± 0.08^a	10.69 ± 0.11^a
Σn-6	97.51 ± 0.56^a	94.04 ± 0.83^c	95.60 ± 0.63^b	87.98 ± 0.05^d	87.85 ± 0.41^d	87.72 ± 0.82^d
MUFA/SFA	0.422 ± 0.001^ab	0.420 ± 0.000^bc	0.422 ± 0.001^a	0.420 ± 0.001^c	0.419 ± 0.001^cd	0.419 ± 0.000^d
PUFA/SFA	0.0741 ± 0.001^b	0.0736 ± 0.001^c	0.0745 ± 0.001^a	0.0698 ± 0.002^d	0.0693 ± 0.001^e	0.0696 ± 0.000^de
Σn-6/Σn-3	9.39 ± 0.01^a	9.30 ± 0.02^b	9.29 ± 0.01^b	8.22 ± 0.03^c	8.17 ± 0.02^d	8.20 ± 0.01^cd

PM: pasteurized milk; MPM: microfiltered and pasteurized milk; SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids; Values were expressed as average ± standard error (SE); Means in same row with same online lowercase letters were not significantly different (p < 0.05).

Levels of short-chain SFAs in PM decreased with increasing in storage days, and they were higher on day 4 than on other days in MPM. The contents of the medium-chain SFAs (except for C13:0) were the highest on day 0 in PM, and the C8:0, C10:0, and C11:0 in MPM had higher concentrations compared with the other storage times. No significant differences (p > 0.05) were found in the concentrations of C12:0, C14:0, and C15:0 in MPM during the whole storage period. PM contained higher (p < 0.05) levels of long-chain SFAs (except for C16:0) compared with the other milks on day 0. There were no significant differences in the contents C16:0, C17:0, C18:0, C22:0, and C24:0 of MPM within 7 days (p > 0.05) suggesting that the five FAs are stable components. Similar phenomenon has been reported by Timmons et al., who confirmed that fatty acid composition in milk was constant during the 8-day storage period.^[31]

The major monounsaturated fatty acids (MUFAs) accumulated in the milks were C18:1n-9, trans-18:1, trans-16:1, and C14:1. For PM, the levels of C18:1n-9 and C14:1 were higher on day 0 than on day 4 and 7, and the contents of 16:1 did not change during the whole storage period. Significant differences (p < 0.05) were found in the concentrations of three different MUFAs (14:1, 16:1, and 18:1n-9) in MPM after storage for 7 days. For polyunsaturated fatty acids (PUFAs), the amounts of cis-18:2n-6, trans-18:2, C18:3n-3, C20:3n-6 and C20:4n-6 were significantly higher than those of C20:3n-3, C20:5n-3, C22:6n-3 and C20:2. Storage periods did not alter the levels of five PUFAs (trans-18:2, C18:2n-6, C20:3n-6, C18:3n-3 and C20:2) in the MF and two PUFAs (C20:2, C20:5n-3) in PM, while the contents of C18:2n-6, C20:3n-6, and C20:4n-6 in MPM were higher on day 0 than on other days. In this study, the total of SFAs in PM on day 0 was the highest among all samples. The period of storage did not have a strong influence on the total SFAs (except for PM on day 0). The content of total UFAs in PM was the highest on day 0, while that of the MPM was lowest on day 7 (p < 0.05).

The ω-3 and ω-6 PUFAs have distinct and opposing physiological properties, and their balance is important for normal growth and development of humans. The content of ω-3 was higher in MPM than in PM, while that of ω-6 was lower in MPM than in PM. Moreover, the storage time did not alter the cholesterol content of MPM. The ω-6/ω-3 ratios in all samples were greater on day 0 than on other days. The ratios of ω-6/ω-3 were lower in MPM than in PM at the same storage time. A redcution in the ratio of ω-6/ω-3 PUFA shows the increased availability of ω-3 PUFAs. The high ratio of ω-6/ω-3 is strongly correlated with the prevalence of cardiovascular diseases and cancer/inflammatory/autoimmune diseases.^[19,29,30] The World Health Organization (WHO) has recommended a ω-6/ω-3 ratio less than 5:1 in total human diet.^[32] In our study, any sample does not meet this criterion due to the nature of milk fat. Thus, according to the ω-6/ω-3 ratio, MPM had healthier properties with respect to PM.

It is worth noting that, compared with PM, the significant decrease in trans isomer formation in MPM over the same storage time could be attributed to the selective removal of microbes or specific enzymes affecting the occurrence of isomerization of oils in raw milk.^[1,33] Generally, lower ratio of ω-6/ω-3 as well as more eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were found in MPM compared with in PM at the same storage period, which demonstrated the beneficial effect of MF on the functional compositions of PM.

CONCLUSIONS

The combination of MF and pasteurization treatment has proved to be an effective tool for improvement of nutrient value and shelf stability of milk. The changes in the distribution of particle size, the contents of cholesterol and CLAs and the profile of FAs in MPM during storage were studied for the first time. Compared with PM, MF treatment enhanced the retention of C18:3n-3, C20:5n-3, C22:6n-3, and Σn-3 FAs, decreased the level of particle size, the contents of cis-9, trans-11 CLAs, and unsaturated FAs as well as the ω-6/ω-3 ratio. The trans-10, cis-12 CLAs, and volumic average diameters did not vary widely at the same storage time among all samples. MF also can cause varying degrees of alteration of individual FAs in milk during storage. The cholestrol content was positively but non-significantly correlated with total fat content, as well as was highly correlated with trans-10, cis-12 CLAs. Significantly positive correlation between the content of cis-9, trans-11 CLAs, and total fat and negative correlation between total fat content and fat globule particle size were observed in milk. Therefore, MF could be proposed as an effective process step during milk production. Future research should focus on the evaluations of bacteriological and bioactive functional parameters of bioactive lipids in MPM. Moreover, the effects of the storage temperature and packaging should be studied.

ACKNOWLEDGMENT

Special thanks to SJTU-Instrumental Analysis Center for expert assistance with the GC experiments.

FUNDING

This research was supported by grants from the Open Project Program of State Key Laboratory of Dairy Biotechnology, Bright Dairy & Food Co. Ltd. (SKLDB2013-02), the Shanghai Minhang District Commission of Science and Technology (2013MH088), the National Science & Technology Pillar Program during the 12th Five-year Plan Period (No. 2013BAD18B02), and the Agricultural Science and Technology Achievements Transformation Fund (2012GB2CO00141).

Additional information

Funding

This research was supported by grants from the Open Project Program of State Key Laboratory of Dairy Biotechnology, Bright Dairy & Food Co. Ltd. (SKLDB2013-02), the Shanghai Minhang District Commission of Science and Technology (2013MH088), the National Science & Technology Pillar Program during the 12th Five-year Plan Period (No. 2013BAD18B02), and the Agricultural Science and Technology Achievements Transformation Fund (2012GB2CO00141).