Simulated in vitro infant gastrointestinal digestion of yak milk fat globules: A comparison with cow milk fat globules

Journal Pre-proofs Simulated in vitro infant gastrointestinal digestion of yak milk fat globules: a comparison with cow milk fat globules Jie Luo, Lu Liu, Tianshu Liu, Qingwu Shen, Chengguo Liu, Hui Zhou, Fazheng Ren PII: DOI: Reference:

S0308-8146(20)30004-2 https://doi.org/10.1016/j.foodchem.2020.126160 FOCH 126160

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

22 July 2019 10 December 2019 2 January 2020

Please cite this article as: Luo, J., Liu, L., Liu, T., Shen, Q., Liu, C., Zhou, H., Ren, F., Simulated in vitro infant gastrointestinal digestion of yak milk fat globules: a comparison with cow milk fat globules, Food Chemistry (2020), doi: https://doi.org/10.1016/j.foodchem.2020.126160

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier Ltd.

Revised manuscript-clear copy

Simulated in vitro infant gastrointestinal digestion of yak milk fat globules: a comparison with cow milk fat globules Running title: Infant gastrointestinal digestion of yak milk fat globules Jie Luoab, Lu Liub, Tianshu Liub, Qingwu Shena, Chengguo Liua, Hui Zhoua and Fazheng Renbc* a College

of Food Science and Technology, Hunan Agricultural University, Changsha,

410114, China b

Key Laboratory of Functional Dairy, Co-constructed by Ministry of Education and

Beijing Government, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China c

Beijing Laboratory of Food Quality and Safety, College of Food Science and

Nutritional Engineering, China Agricultural University, Beijing 100083, China

* Corresponding author: E-mail address: [email protected] Other authors’ E-mail address: Jie Luo: [email protected] Lu Liu: [email protected] Tianshu Liu: [email protected] Qingwu Shen: [email protected] Chengguo Liu: [email protected] Hui Zhou: [email protected]

1

Revised manuscript-clear copy

Abstract Lipolysis products released during digestion exert positive metabolic impacts on the nutrition of newborns. However, the lipolysis behavior of yak milk lipids during digestion remains unknown. In this study, the simulated in vitro infant gastrointestinal digestion of cow, yak and standardized yak milk fat globules the same size as those from cow milk (Cow MF, Yak MF and Yak SMF) were compared. Although Cow MF showed a higher lipolysis rate at the beginning of gastric digestion, Yak MF and Yak SMF exhibited a higher lipolysis level during later gastrointestinal digestion. Higher hydrolysis efficiency of yak milk lipids was due to their lipid properties, including their composition and structure. Furthermore, yak milk lipids released more unsaturated fatty acids than Cow MF throughout digestion. This study highlights the crucial role of lipid characteristics in the efficient digestion of milk lipids and provides new insight for the design of yak milk infant diets. Keywords: Yak milk fat globule; In vitro gastrointestinal digestion, Lipolysis, Infant

2

Revised manuscript-clear copy

1. Introduction Human milk is the gold standard for new born feeding, providing immune modulation, gut maturation and optimal nutrient supply. When human milk is not available, infant formula (IF) is the best substitute for newborns. Fat is the most energy-dense macronutrient in human milk and IFs, delivering about 50% of the energy to an infant (Manson & Weaver, 1997). The fat blends used for IFs are tailored to the composition and structure of human milk fat. Because of the size and interface composition of fat globules, as well as the position of fatty acids (FAs) in cow milk resemble human milk fat to a higher degree than vegetable fats, current research focus is now considered cow milk the main fat source for use in the production of IF products (EFSA Panel on Dietetic Products & Allergies, 2014; Koletzko, 2016). Yak is a bovid species that lives mainly in the Qinghai-Tibetan plateau at heights of 2,500 to 6,000 m above sea level. For centuries, yak dairy products have constituted the base of the Qinghai-Tibetan pastoralists’ daily food intake. Traditionally, Tibetan children drink yak milk from infancy as a substitute for human milk. Additionally, IF products made of yak milk are becoming more popular among consumers outside the Qinghai-Tibetan plateau. According to China dairy industry association, China produces about 1.2 million tons of raw yak milk and 140,000 tons of yak dairy products every year. Among them, the consumption of IF products made of yak milk reached over 4,000 tons in year 2017. Therefore, yak milk lipids could also be used as a fat source in producing IF and follow-on formula. Lipids in milk are present in the form of fat globules enveloped by a biological membrane. The lipolysis products released during lipolysis are well known for their potential positive metabolic impacts on the nutrition of a newborn. For example, cholesterol, milk fat globule membrane (MFGM) phospholipids and long-chain 3

Revised manuscript-clear copy

polyunsaturated fatty acids (LCPUFAs) serve as structural components of cell membranes (Bourlieu, Ménard, Bouzerzour, Mandalari, Macierzanka, Mackie, et al., 2014; Innis, Gilley, & Werker, 2001). Adequate supply of these lipids is particularly important for the development of retinal and brain cells of newborns (Abrahamse, Minekus, van Aken, van de Heijning, Knol, Bartke, et al., 2012). Moreover, sphingolipids such as sphingomyelin (SM) that are present in the MFGM have been shown to play a vital role in gut maturation and myelination of the developing central nervous system of newborns (Spitsberg, 2005). The MFGM is also an exclusive carrier of gangliosides to the neonatal gut, which can be incorporated into the intestinal mucosa and alter membrane fluidity and enterocyte function (Lee, Padhi, Hasegawa, Larke, Parenti, Wang, et al., 2018). Therefore, understanding the digestion fate and kinetics of milk lipids during infant gastrointestinal digestion is important. Gastrointestinal digestion of milk lipids is influenced by the physicochemical characteristics of fat globules, including their size, distribution and composition of the interface, and the FA distribution on the triglyceride backbone (Berton, Rouvellac, Robert, Rousseau, Lopez, & Crenon, 2012). Compared with large fat globules, smaller fat globules have an increased surface area for lipase anchoring that contributes to their faster digestion rate (Singh & Gallier, 2017). In addition to the size of fat globules, the composition of milk lipids also greatly influences their digestion. For example, the adsorption of gastric lipase onto the lipid interface is reinforced when there are more negatively charged phospholipids, such as phosphatidylserine, in the interface membrane (Claire Bourlieu, Paboeuf, Chever, Pezennec, Cavalier, Guyomarc’H, et al., 2016). Moreover, because of the stereospecificity, gastric lipase was found to selectively release short- and medium-chain FAs (Sams, Paume, Giallo, & Carrière, 2015), therefore the FA profile 4

Revised manuscript-clear copy

and its location on the triglyceride backbone also greatly impact the fate of gastrointestinal lipolysis. Compared with cow milk, yak milk fat globules are significantly different, both in their structure and lipid profile. Yak milk fat globules are significantly larger (Luo, Wang, Song, Pang, & Ren, 2016) and contain higher levels of long chain and unsaturated FAs compared with those of cow milk (Ma, He, & Park, 2017). Moreover, in our previous study, we found that yak milk fat globules had interfacial properties distinct from those of cow with higher cholesterol and SM content and a more significant heterogeneous distribution of polar lipids (Luo, Huang, Liu, Zhang, & Ren, 2017). Compositional and structural differences among milk fat globules would influence the extent to which lipids are digested in the gastrointestinal tract and may be physiologically important for the infant. However, the digestibility of yak milk lipids during infant gastrointestinal digestion remains to be elucidated. The objective of this work was to undertake an in vitro study to directly compare the lipid digestion kinetics of yak and cow milk fat globules (the most common animal fat source in IFs) under gastrointestinal conditions that mimic those of infants. To eliminate the effect of globule size, yak milk fat globules with the same average size of those from cow milk were prepared. By comparing the same-sized milk fat globules with different lipid compositions and structures, the relationship between the lipid characteristics and digestion behavior of each type of fat globules was also revealed. 2. Materials and methods 2.1. Materials Bulk raw yak milk from 25 yaks (Bos grunniens) in mid-lactation period was collected from the Treasure of Plateau Yak Dairy Co. LTD. (Ruoergai, Sichuan, 5

Revised manuscript-clear copy

China) in August. Bulk tank raw cow milk was obtained from a local dairy farm with cows mostly in mid-lactation stage (Beijing, China) at the same time. After milking, 0.02% (w/v) sodium azide was immediately added to the milk samples and then stored at 4°C. All milk samples were used for analysis within 3 days. Pepsin powder from porcine gastric mucosa (catalog no. P7000; ≥250 units/mg solid) and fungal lipase from Rhizopus oryzae (50 units/mg solid, stable in the range pH 4.5–7.5, sn-1(3) stereospecific enzyme) were used to simulate the stomach environment. Pancreatin from porcine pancreas (8 × USP specifications), lipase from porcine pancreas (Type II, 100–500 units/mg protein using olive oil) and bile salt extract obtained from Sigma-Aldrich (St. Louis, MO, USA) were used to simulate the intestinal digestion environment. Unless otherwise stated, chemicals were from Sigma-Aldrich. 2.2. Preparation of milk samples Yak milk was first split into two portions. Cow milk and one portion of yak milk were centrifuged at 3,000 × g for 15 min at 4°C, and the top cream layer was collected. To compare the difference between yak and cow milk fat globules in gastrointestinal lipolysis based on the same size of fat globules, another portion of yak milk sample was transferred to a two-stage centrifugation to obtain standardized yak milk fat globules according to a modified method (Lu, Argov-Argaman, Anggrek, Boeren, van Hooijdonk, Vervoort, et al., 2016). Yak milk was first centrifuged at 400 × g for 5 min at 4°C to remove the larger milk fat globule fraction, and the lower layer was collected and then further centrifuged at 3,000 × g for 15 min at 4°C, then the top cream layer was collected as the smaller milk fat globule fraction, namely the standardized yak milk fat globules, which owned the same average size of those from cow milk. The cream layers were all reconstituted to a 3% fat content in simulated 6

Revised manuscript-clear copy

milk ultrafiltrate (SMUF) as the milk fat globules samples, namely Cow milk fat globules (MF), Yak MF and Yak standardized milk fat globules (SMF), respectively. SMUF was prepared according to a previously reported method (Jenness & Koops, 1962). 2.3. Characteristics of the milks and milk fat globules The fat content in the milks and the SMUFs were determined according to Marshall’s method (1992). The particle size distribution of the droplets in the milk samples and SMUF were determined as previously described (Luo, et al., 2017) using a Mastersizer 3000 laser diffraction particle size analyzer (Malvern Instruments Ltd., Malvern, UK). The average volume-weighted diameter D4,3 and the weighted average surface diameter D3,2 were collected. The ζ-potential of fat globules in the milk samples and SMUF were measured using a Malvern Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, UK) as previously described (Luo, et al., 2017). 2.4. Simulated in vitro infant gastrointestinal digestion Gastric digestion. Simulated gastric fluid and digestive enzymes (0.4 mg/mL pepsin and 0.87 mg/mL lipase) were prepared as previously described (Luo, Wang, Li, Chen, Ren, & Guo, 2019) to mimic the physiological processes of infant gastric digestion as reported by Claire Bourlieu, Ménard, De, Sams, Rousseau, Madec, et al. (2015). A pH-stat device (907 Titrando, Metrohm AG, Zurich, Switzerland) was programmed to establish four successive digestion stages at pH 6.5 (0–30 min), pH 6.0 (30–60 min), pH 5.5 (60–90 min) and pH 5.0 (90–120 min). At the beginning of each stage, the sample pH was adjusted to the set value by adding the simulated gastric fluid and the simulated digestion was conducted at 37°C using a continuous stirred system. Intestinal digestion. After 120 min of gastric digestion, the in vitro infant intestinal 7

Revised manuscript-clear copy

digestion was carried out by mixing the digested samples with the simulated intestine solution (1:1), adjusting the pH to 7.0, and adding bile extract (5 mg/mL) (Claire Bourlieu, et al., 2015). Once the digestion started, the mixed enzyme solution (pancreatin and pancreatic lipase, 1.6 mg/mL each) was added and the mixture was incubated at 37°C for 120 min. Release of free fatty acids. The amount of NaOH required for maintaining the equilibrium was used to calculate the concentration of titratable free fatty acids (FFAs) released during the entire simulated in vitro infant gastrointestinal digestion as follows: 𝑀FFA =

𝑉NaOH × 𝑀NaOH msample

where MFFA is the molar amount of FFAs released per milliliter of digested sample (μmol/g), VNaOH is the volume of alkaline solution consumed (mL), MNaOH is the molarity of the alkaline solution (0.2 M), msample is the volume of milk lipid sample digested in the gastrointestinal phase (4.29 mL). Additionally, the degree of lipolysis throughout the gastrointestinal digestion expressed as the percentage of FFA released from the total acyl moieties initially esterified in the triglyceride in the sample was calculated as follows: FFA(%) =

𝑉NaOH × 𝑀NaOH × 𝑀mfTG 𝑊lipid × 2

× 100%

where VNaOH is the volume of alkaline solution consumed (mL), MNaOH is the molarity of the alkaline solution (0.2 M), MmfTG is the average molecular weight of milk fat triacylglycerol deduced from its FA composition (760, 756 and 754 g/mol calculated for Cow MF, Yak MF and Yak SMF, respectively), and Wlipid is the mass of the fat in the assay (3.01 g/100 mL). 2.5. Characterization of milk fat globules during lipid digestion 8

Revised manuscript-clear copy

At various times during both gastric and intestine digestion (0, 30, 60, 90 and 120 min), aliquots (10 mL) were collected and the reactions were terminated by heating at 80°C for 30 min. The samples were then subjected to various analyses. Particle size. Particle size distributions of milk fat globules were determined as described earlier in the text. Morphology observation. The morphologies of milk fat globules during digestion were observed using optical microscopy (Axiovert A1, Zeiss Axioscope, Oberkochen, Germany). Ten microliters of sample was placed on a microscope glass slide, covered with a glass cover slip, and then examined with a 20× magnification lens. Free fatty acid release mode. The initial FA composition (C4:0 to C24:1N9) of the milk samples was analyzed by gas chromatography (GC-14C, Shimadzu, Japan) in the presence of an internal standard (C11) without prior extraction as described in the Chinese Standard (2016). Fatty acid methyl esters (FAMEs) were identified by comparing the relative retention times of the sample FAME peaks with those of the standards (37 component FAME Mix 47885-U; Supelco, Bellafonte, PA, USA). The FFA composition of the milks released during digestion was analyzed after solid phase extraction and compared to the initial FA composition of the milk (esterified total FA) and expressed as mass %. 2.6. Statistical analysis All measurements were performed in triplicate and expressed as the mean ± standard deviation. The digestion data were analyzed by two-way ANOVA with milk group, digestion time and the interaction between group and time as the model variables (with Tukey test) using SPSS software (version 22.0, IBM, Armonk, NY, USA). Other data were analyzed by one-way ANOVA (with Duncan’s multiple range method). The level for statistical significance was set at P < 0.05. 9

Revised manuscript-clear copy

3. Results 3.1. Characteristics of the milk fat globules As shown in Table 1, the fat content, average particle size and absolute ζ-potential value of milk fat globules were all significantly larger for yak milk than cow milk (P < 0.05), which were consistent with our previous studies (Luo, et al., 2016; Luo, et al., 2017). To eliminate the effect of globule size, fat globules were separated from yak milk by two-stage centrifugation separation to permit the selection of droplets with the same average size as those in cow milk. The three types of milk fat globules prepared for the simulated infant gastrointestinal digestion were assigned as fat globules from cow and yak milks (Cow MF and Yak MF) and the standardized fat globules separated from yak milk with the same size of those in cow milk (Yak SMF). As shown in Table 1, the ζ-potential of the Yak SMF was −11.3 ± 0.2 mV, which was not significantly different from that of the original globules from raw yak milk (−11.4 ± 0.4 mV). The results indicated that the fat globules remained undisrupted throughout the centrifugation separation. In contrast, the average volume-weighted diameter D4, 3 of Yak SMF was similar with that of Cow MF (3.05 ± 0.02 vs. 3.03 ± 0.02 μm), indicating the successful standardization of globule size between different species. Furthermore, the fat content of all the milk samples was adjusted to the same level (~3 %), consistent with the fat content in mature human milk (2–6%) (Li, Mu, Jensenevoldthaulov, Xu, Otto, & Anne, 2010). The FA composition of different milk fat globules is presented in Table 2. For all globule types, the major fatty acids were myristate (C14:0), palmitate (C16:0), stearate (C18:0) and oleic acid (C18:1 c9), accounting for approximately 10, 30, 10 and 25% of the total identified fatty acids, respectively. The total saturated and 10

Revised manuscript-clear copy

monounsaturated FA contents were not significantly different among the different milk fat globules (P > 0.05). However, the amounts of short chain fatty acid (SCFA, ≤10 atoms of carbon), medium chain FAs (MCFAs) and polyunsaturated FAs (PUFAs) differed between the species (Table 2). Yak MF contained significantly higher amounts of SCFAs and PUFAs and less MCFAs than did Cow MF (P < 0.05). This is in agreement with the results reported by Teng, Wang, Yang, Ma, & Day (2017). Additionally, the PUFAs and some individual FAs differed between the Yak SMF and Yak MF, which may have potential consequences for digestion. 3.2. FFA release during in vitro infant gastrointestinal lipid digestion 3.2.1 FFA release amount and lipolysis level The development of FFA release from different milk fat globules was first monitored as a function of digestion time (Figure 1). The results indicated significant differences existed in the absolute release amount of FFA as well as the degree of lipid hydrolysis among different species and sizes during gastrointestinal digestion (P < 0.05). During the first 15 min of gastric digestion, the absolute release amount of FFA and the lipolysis rate of the cow milk lipids were higher than those of the yak with a trend of Cow MF ≥ Yak SMF > Yak MF (Figure 1A). However, the lag phase of the Yak SMF hydrolysis occurred after 30 min of gastric digestion, while the amount of FFA released continually increased throughout the entire simulated gastric stage. The amount of FFA released at the end of gastric digestion was of the order of Yak SMF > Yak MF > Cow MF. During the simulated intestinal digestion phase, the amount of FFA released from all fat globules increased steadily toward the end of digestion (Figure 1B). The lipolysis rate and the amount of FFA released from the milk fat globules showed a similar trend as observed for the simulated gastric stage. Yak SMF 11

Revised manuscript-clear copy

exhibited a superior lipid digestibility in the simulated intestinal digestion with a trend of Yak SMF > Cow MF > Yak MF (Figure 1B). Because of the differences in the FA composition and the corresponding differences in milk fat triacylglycerol molecular weights, the degree of lipolysis of milk fat globules based on the initial FFA amount was also calculated throughout the gastrointestinal digestion. As shown in Figure 1C, fat globules from yak milk generally exhibited a higher degrees of lipolysis compared with Cow MF during gastrointestinal digestion. In terms of the function of the globule size, Yak SMF was more easily digested than Yak MF in the simulated gastrointestinal digestion. The degree of lipolysis for Cow MF, Yak MF and Yak SMF determined at the end of gastrointestinal digestion were 81.59±1.49%, 84.85±2.84% and 92.87±3.47%, respectively. 3.2.2 Release mode of free fatty acids To further explore the difference in lipolysis between cow and yak milk lipids, the FFA release mode of different milk fat globules was compared (Figure 2). Consistent with the degree of lipolysis observed in Figure 1C, all types of FFA sharply increased during the early stage of gastric digestion (up to 60 min), and over the intestinal phase. SCFAs rapidly increased during the gastric digestion phase while the release of LCFAs and unsaturated fatty acids (UFAs) was fast during the intestinal digestion because of the stereospecific preference of the gastric lipase and pancreatic lipase, respectively (Sams, et al., 2015). Additionally, the species and globule size impacted the profile of the released FFA. Regardless of the globule size, the fat globules from yak milk showed a significantly higher release rate of UFAs than those of cow milk. The lipolysis of the total UFAs in Cow MF was less than the total SFAs, while an opposite trend was observed for Yak MF and Yak SMF. At the end of the simulated 12

Revised manuscript-clear copy

infant gastrointestinal digestion, 78.63±4.24%, 86.14±0.26% and 96.21±2.33% of UFAs for Cow MF, Yak MF and Yak SMF had been released, respectively. Moreover, Yak SMF generally had a higher FFA release rate than the other samples for all FFA types throughout the gastrointestinal digestion, whereas the curves of Cow MF and Yak MF overlapped during the digestion time (Figure 2). In general, Yak MF had significantly higher release rates of SCFAs during the gastric period and USFA throughout the gastrointestinal digestion. The variations in the cow and yak milk FA release profile might be attributed to the differences in their original fatty acid composition. 3.3 Characteristics of the milk fat globules during digestion 3.3.1 Evolution of particle size distribution We next evaluated the evolution of particle size distribution and the volume-weighted diameter of fat globules during the simulated in vitro infant gastrointestinal digestion, as shown in Figure 3. During the in vitro gastric digestion, the particle size distribution curves of all milk fat globules significantly shifted to the right, indicating that the structure of the fat globules was destabilized, and the globules gradually aggregated. At the beginning of the gastric digestion (within the first 30 min), the fat globule size of all three samples dramatically increased, though the D4,

3

was not different among the globules. However, as the gastric digestion

proceeded (60–120 min), fat globules from yak milk aggregated more than those from the cow milk. The sizes of the Yak MF were distributed over a very wide range and some were even larger than 100 μm. Additionally, the particle size of the Yak SMF remained smaller than the Yak MF throughout the gastric digestion (P < 0.05). During the intestinal digestion period, the aggregated particles were mixed with the simulated intestinal fluid, and the pH was adjusted to 7.0 with bile salt. Upon mixing 13

Revised manuscript-clear copy

with the simulated intestinal fluid, the size of all fat globules immediately decreased, while the Yak SMF showed the greatest decline. As the digestion proceeded, the size of the fat globules gradually decreased as the pancreatic lipase acted, and the shoulder peak became smaller but did not completely return to the distribution observed in the initial raw milk samples. The D4, 3 at the end of the intestinal digestion was in the order of Yak SMF < Cow MF < Yak MF (Figure 3C), with diameters of 7.96 ± 1.1, 13.3 ± 0.9 and 18.1 ± 0.6 μm, respectively. 3.3.2 Microstructural changes To visually observe the lipolysis level during the simulated in vitro infant gastrointestinal digestion of the fat globules, the microstructural changes were monitored by optical microscopy (Figure 4). Before digestion, all milk fat globules were evenly dispersed in the field of vision. After 60 min of gastric digestion, an obvious flocculation and coalescence of fat globules were observed in all samples. The bridging flocculation of the globules could be induced by the association of the lipase with the globules, indicating a gradual loss of stability (Mun, Dekker, & McClements, 2007; Reis et al., 2008). As the gastric digestion proceeded, more fat globules aggregated and coalesced into much larger globules and larger globules were observed in Yak MF compared with the other groups (Figure 4C, H and M). During the intestinal digestion, the flocculation and large-sized coalesced fat globules formed at the gastric phase dissociated to small, uneven particles as shown by the reformed droplets of smaller sizes in Figures 4D, I and N. As digestion proceeded, the amount of large particles and droplets decreased, with a corresponding increase in the amount of small particles. At the end of the intestinal digestion, the sample of Yak SMF did not contain any visibly large aggregates or sediments as determined by visual observation. Some aggregated vesicles were still identified in 14

Revised manuscript-clear copy

the Yak MF sample after 120 min of intestinal digestion. In contrast, though there were almost no aggregates found in the sample of Cow MF, some intact fat globules existed after the intestine phase. 4. Discussion Although cow milk fat is believed to be the main source of fat in IF products, yak milk fat also provides an alternative. Previous results indicated that yak milk fat globules had different composition and structure from those of cow (Luo et al., 2016; Ma et al., 2017), which might affect the lipolysis kinetics during gastrointestinal digestion and may be physiologically important for the infant. This is the first in vitro study to explore the difference in lipolysis kinetics between yak and cow milk lipids in gastrointestinal conditions that mimic those reported in newborns. To eliminate the effect of globule size, standardized fat globules separated from yak milk with the same size of those in cow milk were compared. Therefore, the results of this study allow the determination of the effects of milk species (Cow MF vs. Yak MF), lipid characteristics (Cow MF vs. Yak SMF) and globule sizes (Yak MF vs. Yak SMF) on in vitro infant gastrointestinal lipid digestion. 4.1. Effect of milk species on in vitro infant gastrointestinal lipid digestion Since infants have significantly higher intragastric pH values that are close to the optimal pH of human gastric lipase, gastric digestion plays a central role in lipid digestion in infants (Sams, et al., 2015). At the beginning of gastric digestion, because of the significantly smaller globule size and the corresponding increase in total surface area of Cow MF compared with Yak MF (Table 1), the gastric lipase should act more efficiently on the Cow MF. This may explain why the initial lipolysis rates of the Cow MF were significantly higher than those of the Yak MF. However, the rate of lipolysis of Yak MF was more than Cow MF during the later stage of simulated 15

Revised manuscript-clear copy

gastric digestion. This may have contributed to some compositional and structural advantages, such as higher levels of SCFAs (Table 2) and more SM-rich ordered lipid domains in Yak MF (Luo, et al., 2017). Because of the higher polarity of the SCFAs, the triglycerides consisting of SCFAs have been reported to have more chance to come in contact with lipase and induce a faster lipolysis rate than those consisting of LCFAs (Zhu, Ye, Verrier, & Singh, 2013). Therefore, the higher SCFA in Yak MF compared with Cow MF contributed to the higher lipolysis ability of the Yak MF during the simulated gastric digestion. Furthermore, the ordered lipid domains rich in SM and the mechanical heterogeneity in MFGM are considered to be effective in preferential adsorption and insertion of digestive enzymes (Claire Bourlieu, et al., 2016). Our previous study showed that the presence of lipid domains in yak MFGM was larger in number and wider in size range compared to cow milk (Luo, et al., 2017). Therefore, the lipid domains in yak MFGM could also increase the lipolysis efficiency of Yak MF during the simulated gastric digestion. The size evolution of all milk fat globules was not significantly different within the first 30 min of gastric digestion, but Yak MF had larger particles compared with Cow MF during the later stage of gastrointestinal digestion. This could be the result of a combination of the original particle stability as well as the affinity of the particles toward lipase. Larger size globules and the more dispersed lipid domains in the MFGM of Yak MF (Luo, et al., 2017) might contribute to their lower resistance to deformation and coalescence under lipid digestion than the Cow MF. Furthermore, the higher affinity of the Yak MFGM composition and structure toward the lipase might lead to more globules associating together resulting in globules of larger sizes. When pre-gastric digested milk fat globules enter into the intestine, Yak MF will no longer be preferentially digested over Cow MF. During intestinal digestion, bile salts 16

Revised manuscript-clear copy

may replace some of the original interfacial composition of the fat globules and help to break them down (Singh and Gallier, 2017), which reduces preferential digestion of one species of fat droplet over others. As a result, the original interfacial composition may not dominate the digestion behaviors of the fat globules at longer digestion times. Additionally, because of the interface displacement of the bile salts (Gallier, Ye, & Singh, 2012), and the build-up of fatty acids and monoacylglycerols released from the original globules (Golding & Wooster, 2010), the flocculated globules dissociate as observed by their immediate decrease in size. By the end of the gastrointestinal digestion, some aggregated vesicles were still found in the Yak MF sample. The aggregates observed in this sample may be aggregated lipid droplets or micelles and vesicles assembled from bile salts, phospholipids, FFAs and monoacylglycerols (Maldonado-Valderrama, Wilde, Macierzanka, & Mackie, 2011). In contrast, intact fat particles were still observed in the Cow MF samples. Gallier, Ye, & Singh (2012) and Meena, Rajput, & Sharma (2014) also observed intact fat particles present in the aqueous phase after 120 min of intestinal digestion of cow milk. It should be noted that the intact particles that were observed may not be milk fat globules, but instead were probably mixtures of lipolysis products, such as phospholipids and bile extract, FFAs, and monoacylglycerols (Gallier et al., 2012; J. Li, Ye, Lee, & Singh, 2012; Liang et al., 2017). The particles might also be empty particles that were formed as a result of lipase action leading to the release of FFA and monoacylglycerol (Meena, Rajput, & Sharma, 2014). In regards to the individual FFA release mode between milk species, Yak MF showed significantly higher release rates of SCFAs during the gastric period and UFAs throughout the gastrointestinal digestion, most probably because of the significantly higher percentage of SCFAs and PUFAs in the original Yak MF (Table 17

Revised manuscript-clear copy

2). Our results indicated the profile of FAs liberated after in vitro digestion reflected the patterns found in the corresponding milk sources. Hageman, Keijer, Dalsgaard, Zeper, Carrière, Feitsma, et al. (2019) recently reported that IF containing fat globules from cow milk released more SCFAs (especially C4:0) in the gastric phase of digestion than that of human milk, most probably due to their higher SCFAs percentage in the original globules. Butyrate and other SCFAs are generally considered important for the mediation of beneficial health effect such as intestinal microbiota (Peng, Li, Green, Holzman, & Lin, 2009). Since Yak MF showed an even higher content and release amount of SCFAs than the Cow MF, it is of interest that yak milk has the benefit to make up an infant diet that provides better digestion and absorption of dietary lipids. Furthermore, human milk fat contains about 10-fold linoleic acid content than that in the cow milk (Hageman, Danielsen, Nieuwenhuizen, Feitsma, & Dalsgaard, 2019), and the lipolysis of human milk results in higher levels of UFAs compared to the cow milk (Hageman et al., 2019). Therefore, the higher UFAs released from Yak MF than the Cow MF would greatly benefit the in vivo development of the retinal and brain cells of infants (Poquet & Wooster, 2016). 4.2. Effect of lipid characteristics on in vitro infant gastrointestinal lipid digestion For fat globules with the same sizes, differences in the rate and extent of digestion can be attributed to the different fatty acid compositions of triglycerides in milk fat, surface layers and their stereospecific locations. The comparison between Yak SMF and Cow MF based on the same globule size described herein complements the above results. Current results indicated that Yak SMF was more efficiently digested than Cow MF throughout the entire gastrointestinal digestion as supported by its higher degree of lipolysis, increased amount of FFAs released and smaller particle size. 18

Revised manuscript-clear copy

During the gastric digestion, the lipolysis of Yak SMF was 9.13% higher than that of Cow MF. In terms of the intestinal phase of digestion, the advantage was gradually narrowed because of the substitution effect of the bile salts, but the FFA lipolysis of the Yak SMF was still about 2.14% higher than that of the Cow MF. In addition to the FFA composition and MFGM lipid structural advantages of fat globules from yak milk over those of cow milk as discussed above, the location of the FAs in the triglycerides might also contribute to the better digestibility of Yak SMF. Gastric and pancreatic lipases show strong regiospecificity by hydrolyzing the sn-1 and sn-3 positions of the triglyceride to generate sn-1, 2 (2, 3) diglycerides, sn-(2) monoglycerides and FFA (Armand, 2008). In contrast, SCFAs and long chain unsaturated fatty acids are predominantly located at the sn-1 and sn-3 positions in milk fat whereas MCFAs and long chain saturated fatty acids are predominantly found at the sn-2 position (Kalo, Kemppinen, Ollilainen, & Kuksis, 2004). Furthermore, as lipolysis proceeds, there is a build-up of 2-monoglycerides and FFAs on the surfaces of the fat globules, which can limit lipase adsorption to these surfaces (Golding et al., 2010). Long chain fatty acids (LCFAs) and their 2-monoglycerides have considerably higher interfacial activity than SCFAs and their 2-monoglycerides (Reis, Miller, Leser, Fainerman, & Holmberg, 2008). When compared to human milk, there is a higher percentage of LCSFAs placed at the sn-1 and sn-3 position of the triglyceride in cow milk, especially the palmitic acid (Hageman, et al., 2019). Given the fact that Yak SMF and Cow MF have the same LCFA content (as shown in Table 2), if there were more LCFAs located in the sn-1 and sn-3 positions of Cow MF than the Yak SMF, more lipolysis products should accumulate on the interface of the fat globules and partly protect them from lipolysis in the gastric phase of digestion. Therefore, the better digestibility of Yak SMF than the Cow MF may be attribute to a 19

Revised manuscript-clear copy

more similar distribution of LCSFAs in the triglyceride core in yak milk lipids to that of the human milk. Additionally, as shown in Figure 2, the amount of MCFAs released from Yak SMF was significantly higher than that of the Cow MF, although its MCFA content was significantly lower (Table 2). Thus, the higher lipolysis level of Yak SMF might also be attributed to the higher level of MCFAs at the sn-1 or sn-3 positions of the triglyceride. Therefore, the stereospecific location of the FAs in the yak triglycerides may also be an important factor that increases the lipolysis rate of the Yak SMF. However, this remains speculative and further study is necessary to clarify this. It should be noted that by comparing Yak SMF, Yak MF and Cow MF, the lipolysis advantage of Yak SMF over Cow MF was larger than that between Yak MF and Cow MF. The results indicated that fat globules from yak milk were more efficiently digested than the Cow MF mainly because of their lipid characteristics (composition and location), which outweigh the disadvantages brought about by their larger size. Thus, the lipid characteristics played a more dominant role in the gastrointestinal lipid digestion of infants when compared with the globule droplet size. 4.3. Effect of globule size on in vitro infant gastrointestinal lipid digestion Consistent with the results from previous studies (Berton, et al., 2012; Garcia, Antona, Robert, Lopez, & Armand, 2014), the digestion and FFA release patterns of Yak MF and Yak SMF suggested that varying the sizes of the milk fat globules resulted in distinct digestion behaviors. Smaller globules provide more total surface area for lipase action compared with larger globules, which leads to their higher digestibility (Berton, et al., 2012). What should be noted is that the fat globules of different sizes studied herein not only varied in size, but also slightly varied in their 20

Revised manuscript-clear copy

lipid composition. Compared with the Yak MF, the Yak SMF had a different FA profile, especially in its PUFA content (Table 2). Additionally, although not determined in the present study, the MFGM composition and location might also differ among yak milk fat globules of different sizes, owing to the widely recognized fact that small and large cow milk fat globules differ in their lipid characteristics (Fauquant, Briard, Leconte, & Michalski, 2005; Mesilati-Stahy, Mida, & Argov-Argaman, 2011). Therefore, besides the size effect, the differences in lipid properties, including composition, structure and distribution, might also exert a non-ignorable influence on the lipolysis ability observed between Yak MF and Yak SMF. 5. Conclusion Previous studies revealed that yak and cow milk fat globules differ in terms of their structure and lipid profiles. The current study complements previous studies by revealing the differences in lipolysis behavior of fat globules from yak and cow milk during simulated infant gastrointestinal digestion for the first time. In general, fat globules from yak milk were more efficiently digested compared with those from cow milk during the in vitro infant gastrointestinal digestion process. This superior lipolysis ability was mainly attributed to the lipid characteristics of the yak milk fat globules, which could even outweigh the disadvantages brought about by their larger size. The specific composition and location of the triglyceride core and the interfacial layer surrounding the fat globules might play a more critical role in the lipolysis in the digestive tract of infants when compared with the droplet size. Furthermore, fat globules from yak milk showed significant higher release rates of SCFAs during gastric digestion and higher UFAs throughout gastrointestinal digestion, which could be a great benefit for the development of infants. An improved understanding of the 21

Revised manuscript-clear copy

digestive characteristics of yak milk lipids during infant gastrointestinal digestion should be useful for the development of an infant diet that provides better digestion and absorption of dietary lipids by infants. The stereospecific location of the FAs in the human, cow and yak milk triglycerides and effect of technological treatments on yak milk lipid digestion would be worthy of further study. Acknowledgments Finance support was provided by National Natural Science Foundation of China (31901717) and National Key R&D Program of China (No. 2017YFD0400605). We thank Renee Mosi, PhD, from Liwen Bianji, for editing the English text of a draft of this manuscript. Reference Abrahamse, E., Minekus, M., van Aken, G. A., van de Heijning, B., Knol, J., Bartke, N., Oozeer, R., van der Beek, E. M., & Ludwig, T. (2012). Development of the digestive system—experimental challenges and approaches of infant lipid digestion. Food Digestion, 3(1-3), 63-77. Armand, M. (2008). Milk fat digestibility. Sciences Des Aliments, 28(1-2), 84-98. Berton, A., Rouvellac, S., Robert, B., Rousseau, F., Lopez, C., & Crenon, I. (2012). Effect of the size and interface composition of milk fat globules on their in vitro digestion by the human pancreatic lipase: Native versus homogenized milk fat globules. Food Hydrocolloids, 29(1), 123-134. Bourlieu, C., Ménard, O., Bouzerzour, K., Mandalari, G., Macierzanka, A., Mackie, A. R., & Dupont, D. (2014). Specificity of infant digestive conditions: some clues for developing relevant in vitro models. Critical Reviews in Food Science & Nutrition, 54(11), 1427.

22

Revised manuscript-clear copy

Bourlieu, C., Ménard, O., De, L. C. A., Sams, L., Rousseau, F., Madec, M. N., Robert, B., Deglaire, A., Pezennec, S., & Bouhallab, S. (2015). The structure of infant formulas impacts their lipolysis, proteolysis and disintegration during in vitro gastric digestion. Food Chemistry, 182(39), 224-235. Bourlieu, C., Paboeuf, G., Chever, S., Pezennec, S., Cavalier, J. F., Guyomarc’H, F., Deglaire, A., Bouhallab, S., Dupont, D., & Carrière, F. (2016). Adsorption of gastric lipase onto multicomponent model lipid monolayers with phase separation. Colloids Surf B Biointerfaces, 143, 97-106. EFSA Panel on Dietetic Products, N., & Allergies. (2014). Scientific Opinion on the essential composition of infant and follow‐on formulae. EFSA Journal, 12(7), 3760. Fauquant, C., Briard, V. r., Leconte, N., & Michalski, M.-C. (2005). Differently sized native milk fat globules separated by microfiltration: fatty acid composition of the milk fat globule membrane and triglyceride core. European Journal of Lipid Science and Technology, 107(2), 80-86. Gallier, S., Ye, A., & Singh, H. (2012). Structural changes of bovine milk fat globules during in vitro digestion. Journal of Dairy Science, 95(7), 3579-3592. Garcia, C., Antona, C., Robert, B., Lopez, C., & Armand, M. (2014). The size and interfacial composition of milk fat globules are key factors controlling triglycerides bioavailability in simulated human gastroduodenal digestion. Food Hydrocolloids, 35(1), 494-504. Golding, M., & Wooster, T. J. (2010). The influence of emulsion structure and stability on lipid digestion. Current Opinion in Colloid & Interface Science, 15(1–2), 90-101.

23

Revised manuscript-clear copy

Hageman, J. H., Keijer, J., Dalsgaard, T. K., Zeper, L. W., Carrière, F., Feitsma, A. L., & Nieuwenhuizen, A. G. (2019). Free fatty acid release from vegetable and bovine milk fat-based infant formulas and human milk during two-phase in vitro digestion. Food & function, 10(4), 2102-2113. Hageman, J. H., Danielsen, M., Nieuwenhuizen, A. G., Feitsma, A. L., & Dalsgaard, T. K. (2019). Comparison of bovine milk fat and vegetable fat for infant formula: Implications for infant health. International Dairy Journal, 92, 37-49. Innis, S. M., Gilley, J., & Werker, J. (2001). Are human milk long-chain polyunsaturated fatty acids related to visual and neural development in breast-fed term infants? The Journal of pediatrics, 139(4), 532-538. Jenness, R., & Koops, J. (1962). Preparation and properties of a salt solution which simulates

milk

ultrafiltrate.

NEDERLANDS

MELK-EN

ZUIVELTIJDSCHRIFT, 16(3), 153-164. Kalo, P., Kemppinen, A., Ollilainen, V., & Kuksis, A. (2004). Regiospecific determination of short‐chain triacylglycerols in butterfat by normal‐phase HPLC with on-line electrospray-tandem mass spectrometry. Lipids, 39(9), 915-928. Koletzko, B. (2016). Human milk lipids. Annals of Nutrition and Metabolism, 69(Suppl. 2), 27-40. Lee, H., Padhi, E., Hasegawa, Y., Larke, J., Parenti, M., Wang, A., Hernell, O., Lönnerdal, B., & Slupsky, C. (2018). Compositional dynamics of the milk fat globule and its role in infant development. Frontiers in pediatrics, 6, 1-20.

24

Revised manuscript-clear copy

Li, J., Ye, A., Lee, S. J., & Singh, H. (2012). Influence of gastric digestive reaction on subsequent in vitro intestinal digestion of sodium caseinate-stabilized emulsions. Food & function, 3(3), 320-326. Li, Y., Mu, H., Jensenevoldthaulov, A., Xu, X., Otto, M., & Anne, O. (2010). New human milk fat substitutes from butterfat to improve fat absorption. Food Research International, 43(3), 739-744. Lu, J., Argov-Argaman, N., Anggrek, J., Boeren, S., van Hooijdonk, T., Vervoort, J., & Hettinga, K. A. (2016). The protein and lipid composition of the membrane of milk fat globules depends on their size. Journal of Dairy Science, 99(6), 4726-4738. Luo, J., Huang, Z., Liu, H., Zhang, Y., & Ren, F. (2017). Yak milk fat globules from the Qinghai-Tibetan Plateau: membrane lipid composition and morphological properties. Food Chemistry. Luo, J., Wang, Z.-w., Song, J.-h., Pang, R.-p., & Ren, F.-z. (2016). Lipid Composition of Different Breeds of Milk Fat Globules by Confocal Raman Microscopy. Spectroscopy and Spectral Analysis, 36(1), 125-129. Luo, J., Wang, Z., Li, Y., Chen, C., Ren, F., & Guo, H. (2019). The simulated in vitro infant gastrointestinal digestion of droplets covered with milk fat globule membrane polar lipids concentrate. Journal of Dairy Science. Ma, Y., He, S., & Park, Y. W. (2017). Yak milk. Handbook of Milk of Non-Bovine Mammals. Maldonado-Valderrama, J., Wilde, P., Macierzanka, A., & Mackie, A. (2011). The role of bile salts in digestion. Advances in Colloid & Interface Science, 165(1), 36-46.

25

Revised manuscript-clear copy

Manson, W. G., & Weaver, L. T. (1997). Fat digestion in the neonate. Archives of Disease in Childhood-Fetal and Neonatal Edition, 76(3), F206-F211. Marshall, R. T. (1992). Standard Methods for the Examination of Dairy Products. 16th ed. American Public Health Association, Washington, DC. Meena, S., Rajput, Y., & Sharma, R. (2014). Comparative fat digestibility of goat, camel, cow and buffalo milk. International Dairy Journal, 35(2), 153-156. Mesilati-Stahy, R., Mida, K., & Argov-Argaman, N. (2011). Size-Dependent Lipid Content of Bovine Milk Fat Globule and Membrane Phospholipids. Journal of Agricultural and Food Chemistry, 59(13), 7427-7435. Peng, L., Li, Z.-R., Green, R. S., Holzman, I. R., & Lin, J. (2009). Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers. The Journal of nutrition, 139(9), 1619-1625. Poquet, L., & Wooster, T. J. (2016). Infant digestion physiology and the relevance of in vitro biochemical models to test infant formula lipid digestion. Molecular nutrition & food research, 60(8), 1876-1895. Sams, L., Paume, J., Giallo, J., & Carrière, F. (2015). Relevant pH and lipase for in vitro models of gastric digestion. Food & function, 7(1), 30. Singh, H., & Gallier, S. (2017). Nature's complex emulsion: The fat globules of milk. Food Hydrocolloids, 68, 81-89. Spitsberg, V. (2005). Invited review: Bovine milk fat globule membrane as a potential nutraceutical. Journal of Dairy Science, 88(7), 2289-2294. Teng, F., Wang, P., Yang, L., Ma, Y., & Day, L. (2017). Quantification of fatty acids in human, cow, buffalo, goat, yak, and camel milk using an improved one-step GC-FID method. Food Analytical Methods, 10(8), 2881-2891. 26

Revised manuscript-clear copy

Zhu, X., Ye, A., Verrier, T., & Singh, H. (2013). Free fatty acid profiles of emulsified lipids during in vitro digestion with pancreatic lipase. Food Chemistry, 139(1-4), 398-404. Fazheng Ren conceived and designed the experiments; Lu Liu and Tianshu Liu performed the experiments; Jie Luo and Qingwu Shen analyzed the data; Chengguo Liu and Hui Zhou contributed analysis tools and provided scientific guidance; Jie Luo wrote the paper. All authors read and approved the manuscript.

Figure 1. Total free fatty acid (FFA) release amount of different milk fat globules during in vitro (A) Gastric digestion, (B) Intestinal digestion and the corresponding degree of lipolysis (C, D) during the in vitro gastrointestinal phase. Yak MF and Cow MF: fat globules separated from yak and cow milk, respectively. Yak SMF: standardized fat globules separated from yak milk with the same size of those in cow milk. Error bars represent the standard deviation (SD) of triplicate experiments. Data in (D) are shown as the mean ± SD, n=3. Different superscript letters in the same column indicate significant differences among samples; P < 0.05. Figure 2. Free fatty acids (FFA) released percentages during in vitro infant gastrointestinal digestion. (A) Short chain fatty acids (SCFAs, chain length from 4 to 10 C) released; (B) Medium chain fatty acids (MCFAs, chain length from 12 to 15 C) released; (C) Long chain fatty acids (LCFAs, chain length from 16 to 24 C) released; (D) Saturated fatty acids (SFAs) released; (E) Unsaturated fatty acids (UFAs) released. Yak MF and Cow MF: fat globules separated from yak and cow milk, respectively. Yak SMF: standardized fat globules separated from yak milk with the same size of

27

Revised manuscript-clear copy

those in cow milk. Error bars represent the standard deviation of triplicate experiments. Figure 3. (A) Evolution of particle size distribution and volume-weighted diameter of different milk fat globules during in vitro infant (B) gastric (GAS) and (C) intestinal (INT) digestion. Yak MF and Cow MF: fat globules separated from yak and cow milk, respectively. Yak SMF: standardized fat globules separated from yak milk with the same size of those in cow milk. Error bars represent the standard deviation of triplicate experiments. Figure 4. Morphologies of different milk fat globules during in vitro infant gastric (GAS) and intestinal (INT) digestion: (A-E) Fat globules separated from cow milk (Cow MF), (F-J) fat globules separated from yak milk (Yak MF) and (K-O) standardized fat globules separated from yak milk with the same size of those in cow milk (Yak SMF). Morphologies of different milk fat globules before in vitro digestion were taken as controls (A, F, and K).

28

Revised manuscript-clear copy

Table 1. Physicochemical characteristics of milks and the fat globules1

29

Revised manuscript-clear copy

Yak milk

Cow milk

Yak MF2

Cow MF

Fat (g.100mL-1) 5.25 ± 0.04 a 3.26 ± 0.02b 3.02 ± 0.02c 3.01 ± 0.01c

1

Yak SMF 3.01 ± 0.02c

D4, 3 (μm)

4.57± 0.02a

3.03 ± 0.01b 4.58 ± 0.01a 3.03 ± 0.02b 3.05 ± 0.02b

D3, 2 (μm)

2.88 ± 0.01a

2.39 ± 0.01b 2.91 ± 0.01a 2.39 ± 0.01b 2.31 ± 0.01c

ζ (mV)

-11.4 ± 0.4a

-10.9 ± 0.2a

-11.5 ± 0.2b

-10.7 ± 0.3c

-11.3 ± 0.2b

Mean ± standard deviation, n=3. Different superscript letters in the same line

indicate significant differences within group; P < 0.05. 2

Yak MF and Cow MF: fat globules separated from yak and cow milk and dispersed

in SMUF, respectively. Yak SMF: standardized fat globules separated from yak milk with the same size of those in cow milk.

30

Revised manuscript-clear copy

Table 2. Fatty acid compositions of different milk fat globules1 Fractions (%)

Fatty acids Cow MF2

Yak MF

Yak SMF

C4:0

1.54 ± 0.12b

2.28 ± 0.07a

2.55 ± 0.11a

C6:0

1.25 ± 0.18b

1.89 ± 0.07a

1.47 ± 0.12b

C8:0

0.65 ± 0.03b

0.95 ± 0.04b

1.21 ± 0.07a

C10:0

2.01 ± 0.14a

2.21 ± 0.09a

2.03 ± 0.08a

C12:0

3.14 ± 0.10a

2.08 ± 0.15b

2.24 ± 0.13b

C14:0

11.52 ± 0.25a

8.63 ± 1.09b

8.29 ± 0.32b

C14:1

1.22 ± 0.17a

0.42 ± 0.07b

0.53 ± 0.06b

C15:0

0.93 ± 0.07c

1.56 ± 0.03a

1.29 ± 0.13b

C15:1

0.22 ± 0.01b

0.31 ± 0.07a

0.33 ± 0.04a

C16:0

33.61 ± 2.03a

31.42 ± 0.70a

31.68 ± 1.21a

C16:1

2.71 ± 0.02a

2.14 ± 0.07b

2.64 ± 0.07a

C17:0

0.65 ± 0.03c

1.03 ± 0.05a

0.89 ± 0.03b

C17:1 c10

0.21 ± 0.01c

0.24 ± 0.01b

0.26 ± 0.01a

C18:0

10.26 ± 0.38b

12.61 ± 0.27a

12.08 ± 0.52a

C18:1 c9

25.91 ± 1.17b

27.75 ± 1.57a

27.02 ± 1.00a

C18:1 t9

2.33 ± 0.14ab

2.07 ± 0.20b

2.42 ± 0.31a

C18:2 c9,12

1.33 ± 0.07c

1.41 ± 0.09c

1.78 ± 0.06a

C18:3 n3

0.28 ± 0.03c

0.72 ± 0.01b

0.96 ± 0.03a

C20:4

0.23 ± 0.01c

0.28 ± 0.01b

0.33 ± 0.03a

SCFA

5.45 ± 0.37b

7.33 ± 0.27a

7.26 ± 0.35a

31

Revised manuscript-clear copy

1

MCFA

17.03 ± 2.69a

13.00 ± 1.20b

12.68 ± 2.02b

LCFA

77.52 ± 2.09a

79.67 ± 2.33a

80.06 ± 2.08a

SFA

65.56 ± 3.23a

64.66 ± 1.52a

63.73 ± 2.47a

MUFA

32.60 ± 1.81a

32.93± 2.17a

33.20± 1.88a

PUFA

1.84 ± 0.11c

2.41 ± 0.31b

3.07± 0.10a

Mean ± standard deviation, n=3. Different superscript letters in the same line

indicate significant differences within group; P < 0.05. 2

Abbreviations: Yak MF and Cow MF: fat globules separated from yak and cow

milk and dispersed in SMUF, respectively; Yak SMF: standardized fat globules separated from yak milk with the same size of those in cow milk; SCFA: short chain fatty acids (chain length from 4 to 10 C); MCFA: medium chain fatty acids (chain length from 12 to 15 C); LCFA: long chain fatty acids (chain length from 16 to 24 C); SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.



Yak milk fat globules were subjected to simulated in vitro infant digestion



Yak milk fat globules were generally more efficiently hydrolyzed than cow milk fat



Yak milk fat globules released more unsaturated fatty acids than cow milk fat



The superior lipid digestion of yak milk lipids was attributed to their lipid properties

32