Dietary strategies to enrich milk with healthy fatty acids – a review


 Feed is the main factor impacting the composition and quality of milk of dairy animals. Therefore, the present review explores the effects of feed and nutrition on milk fat content and levels of healthy fatty acids (FA) in milk consumed by humans. Milk and dairy products are two main sources of healthy and unhealthy FA in human nutrition. The concentrations of FA in milk depend mainly on diets; therefore, milk FA concentrations and ratios can be greatly altered by some feeding strategies. Dietary supplementation of the diets of dairy livestock with vegetable seeds or oils, microalgae and phytogenic feed additives, and feeding of some grasses can enhance the contents of healthy FA, including n-3 FA, α-linolenic acid, conjugated linoleic acid (CLA) and, generally, unsaturated FA in milk and dairy products. Enrichment of milk with healthy FA may make milk a source of anticarcenogens (CLA and polyphenols) for human health. This review, therefore, focusses on the current research findings on enrichment of milk with healthy FA and summarizes some effective supplementation strategies to alter milk FA profile.

4 rich microalgae, phytogenic feed additives, and other feed supplements on milk FA profile are discussed in detail. For purpose of this review, feed additive refers to non-nutritive substance added to feed in micro quantities to enhance feed utilization efficiency and improve animal performance, while feed supplement is used to describe additional nutritive materials, containing phytogenic substances/secondary metabolites, given to animals to enhance feed utilization.

Milk fat, fatty acids, and biohydrogenation
Fat in milk occurs in form of globules of varying sizes, with the triglycerides enclosed in a triple-layer membrane. The fat globules (i.e., diameter) number and size depend on such factors as physiology, environment and genetics. Triacylglycerols, accounting for more than 98% of lipids in livestock milk, comprise glycerol and three FA with diverse carbon chain lengths (Nudda et al., 2014). Milk FA are synthesised and secreted by the mammary gland epithelial cells, using plasma uptake or de novo synthesis as the main source. Ruminal acetate and beta-hydroxybuyrate obtained during fermentation are the sources of the de novo synthesised milk FA. The de novo synthesis of FA produces all the short-chain FA (C4:0-C14:0) and part of C16:0, while the remaining part of C16:0 and virtually all of the milk longchain FA (C18:0-C22:0) emanate from circulating blood lipids and absorption in the small intestine or mobilization of adipose tissue (Nudda et al., 2014). However, the activity of desaturase enzymes may further modify the C14:0-C18:0 FA in the mammary gland.
Additionally, odd and branched-chain FA in milk fat are obtained largely from the intestinal absorption of lipids from the bacteria membrane coming from the rumen (Vlaeminck et al., 2006).
Milk fat content and composition depend mainly on the diet of animals and can be dramatically changed by altering ruminal fermentation, especially acetate and acetate/propionate ratio (El-Zaiat et al., 2019;Azzaz et al., 2020). The proportions of ruminal acetate, butyrate, and propionate depend on dietary neutral detergent fibre (NDF) and non-fibre carbohydrates (NFC). Cannas (2009) recommended NDF ranging from 33% to 45% and NFC ranging from 28 to 38% of the diet as levels for optimal ruminal function, milk production, and fat concentration. Moreover, milk fat content and composition greatly depend on the energy balance of animal, especially in early lactation. Negative energy balance increases milk fat level and its content of long-chain-preformed FA as a result of the absorption of non-esterified FA obtained from mobilization of body fat in the mammary gland (Pulina et al., 2006). 5 Feeds and pattern of ruminal fermentation are responsible for milk FA variation.
Production of trans FA isomers in the rumen causes depression of milk fat, and ruminal biohydrogenation of FA provides a clue to the source of specific trans FA isomers used by the mammary enzymes for secretion in milk (Tripathi, 2015). The ability of ruminal microbial biohydrogenation, which transforms the UFA of dietary fat to saturated FA, to change milk FA composition irrespective of the dietary FA composition is limited. This, therefore, limits the transfer of UFA to mammary tissue even at high dietary concentration. Ruminal microflora are responsible for biohydrogenation, a process involving addition of hydrogen through microbial enzymes and elimination of double bonds in a fatty acyl chain and their conversion from UFA to SFA. The roles played by ruminal microorganisms in regulating pathway of FA biohydrogenation for successful nutritional manipulation of milk constituents are imperative.
Trans FA and milk fat depression are the consequences of the intermediates of trans FA, which arise from the biohydrogenation pathways. Conjugated linoleic acid, an essential biohydrogenation intermediates in milk fat, is beneficial to human health due to its anticarcinogenic properties. Similarly, the cis-9, trans-11 CLA isomer, which arises from linoleic acid biohydrogenation, possesses special anticarcinogenic properties. The process of improving milk biohydrogenation intermediates, which involve knowing the origin and probable enhancement of ruminal beneficial FA isomers, was identified by a multitude of positional and geometric trans isomers produced from ruminal lipid biohydrogenation. Greater than 10 positional isomers of trans monoene FA and a dozen or more CLA isomers have been isolated in the contents of ruminants intestine. Biohydrogenation of linolenic and linoleic acids produced a trans -10 double bond intermediate. Ruminal bacteria can produce a trans -10, cis -12 CLA, while mixed rumen microorganisms can convert oleic acid to trans FA and also produce a trans -10 isomer (Tripathi, 2015).

Enrichment of milk with healthy FA
Many feeding strategies such as the amount and types of feed supplement, especially secondary metabolites containing forage, and addition of vegetable or marine oils and algae to diets have been shown to cause major variation in milk FA composition because of the effect of these factors on the process of ruminal biohydrogenation of dietary UFA (Nudda et al., 2014;Gebreyowhans et al., 2019). Table 1 summarizes the effect of pasture-based diets on milk FA profile. In their review, Kalač and Samková (2010) showed that grasses are the main and cheapest source of FA in ruminant diet, though they contain relatively low (2 to 5% DM) total FA concentration. Some feed supplements, like green pasture, are an excellent source of ALA fatty acid, presenting about 50-75% of the total FA (Chilliard et al., 2007). The ALA is considered as one of the most effective FA affecting milk FA composition and contributing to increased healthy FA (Dewhurst and Moloney, 2013;Nudda et al., 2014;de la Torre-Santos et al., 2020). The green pasture ALA is partially biohydrogenated in the rumen to vaccenic acid, released into milk, and partly transformed to cis-9, trans-11 CLA by the action of stearoyl-CoA desaturase in the mammary tissue (Nudda et al., 2014). De Renobales et al. (2012) observed strong relationships between green pasture intake and ALA milk content (R 2 = 0.69) and CLA (R 2 = 0.79). de La Torre-Santos et al. (2020) compared the effect of mode of grass provision (grazing, zerograzing, or ensiling) on the performance of dairy cows and observed that cows on grazing treatment had greater proportions of vaccenic and rumenic acids, and C18:1 trans-11/C18:1 trans-10 ratio compared with cows on zero-grazing and grass silage treatments. Additionally, they observed increased proportion of linoleic acid for the grazing treatment, while treatments had no effect on ALA, SFA, UFA, or n-6/n-3 ratios.

Effects of pasture-based diets
The species of pasture affects milk FA profile (Nudda et al., 2014). Addis et al. (2005) observed that the milk of sheep fed legume-based pastures, as a feed supplement, showed greater CLA and ALA contents and lower content of SFA relative to the milk of sheep fed ryegrass pasture. They also observed that the intake of crown daisy (Chrysanthemum coronarium L.) and sulla (Hedysarum coronarium L.) favoured greater level of CLA in milk fat. Kliem et al. (2008) noted that maize silage replacement for grass silage decreased milk total n-3 FA, ALA, and EPA, and improved n-6/n-3 ratio. In addition to increasing milk ALA content, increased EPA and DHA contents were obtained from cows kept under grass-based diet (Kalač and Samková, 2010).
The physical form of pasture can also affect milk FA profile. Mohammed et al. (2009) observed improved milk ALA content with fresh grasses compared with conserved grasses. Cabiddu et al. (2006) attributed pronounced effect of forage species on cheese FA composition to varied feed FA composition and intake. In many experiments, grass-based diet increased ALA content from 0.10 to 0.33% of FA and decreased SFA content from 6.66 to 2.00% of FA in the milk of ruminants compared with total mixed ration-based feeding (Kalač and Samková, 2010;Rego et al., 2016). Table 2  (2015) did not observe any differences between the effect of sunflower oil or seeds on milk FA proportions. Oil inside intact seeds is released gradually whereas oil extracted from the seed is immediately available in the rumen.

Effect of plant seeds and oils
Processed dietary fats (e.g., extruded, rolled, micronized, or roasted seeds) generally effectively increase CLA content of milk compared to raw seeds (Kliem et al., 2019). This is 8 possibly because of slow and complete release of the oil content of raw seeds in the rumen relative to the processed seeds, which have a reduced effect on the ruminal environment. Kliem et al. (2019) showed that extruded linseed oil supplementation to lactating cows increased ALA and total n-3 FA contents and decreased total SFA content and n-6/n-3 ratio. Others (Suksombat et al., 2016) observed improvements in milk ALA and total n-3 FA contents and reductions in milk total SFA concentration and n-6/n-3 ratio with un-extruded linseed oil or whole linseed supplementation to lactating cows.

Effect of microalgae and marine oil
Generally, ruminant feeds contain low amounts of DHA and EPA, which are, therefore, marginally present or totally absent in dairy products. In contrast, microalgae are rich in DHA and EPA (Gomaa et al., 2018) and are thus used as a good source of these FA in the diets of dairy animals to produce dairy products with enhanced concentrations of DHA and EPA (Lum et al., 2013;Altomonte et al., 2018).
Increased concentrations of milk DHA (from 1000 to 1122%) and EPA (from 24 to 240%) have been observed in cows supplemented with full-fat microalgae biomass (Moran et al., 2018). The transformation of DHA and EPA from feed to milk is more efficient with full-fat microalgae compared with the defatted microalgae (Gebreyowhans et al., 2019). However, Lum et al. (2013), in their review, stated that feeding of defatted microalgae biomass to dairy cows increased the DHA and EPA and decreased the SFA concentrations in milk. Till et al.
(2020) noted that supplementation of dairy cow diet with Schizochytrium limacinum microalgae increased milk fat content of C22:6n-3, total PUFA, and total n-3 PUFA but reduced n-6/n-3 PUFA ratio. Though marine oil is poor in CLA precursors, its supplementation increased milk vaccenic acid and CLA through the inhibition of the reduction of vaccenic acid to C18:0 by rumen bacteria. Mozzon et al. (2002) supplemented diets of ewes with protected fish oil at 30 and 45 g/d and observed increased milk CLA content. Studies (Mozzon et al., 2002) on the effect of supplementing the diet of ewes with rumen-protected marine oil rich in EPA and DHA reported increased concentrations of these long-chain FA in milk of the supplemented ewes relative to the milk of the control ewes.  From the previous literature, FA of small ruminant milk was more sensitive to essential oils and phytogenic supplementation compared to the FA of cow milk, suggesting that essential oil could mitigate biohydrogenation process and potentially improve milk nutraceutical. This variation in the FA of small and large ruminants milk could be due to the rapid/faster ruminal passage rate of ingesta in the small ruminants relative to the large ruminants, which possibly restrict rumen bacteria ability or their need to complete biohydrogenation process (Nudda et al., 2014;Abd El Tawab et al., 2020). Table 5   Technol. 131, 389-417. Table 1. Effect of pasture-based diets on milk fatty acid profile ↑ = increased, ↓= decreased, l = no effect.  ↑ cis-9, cis-12, cis-9, trans-11 C18:2, C20:3n-3, C20:3n-6, C20:5n-3 and C22:6n-3.

Effect of altering milk FA on oxidative stability and sensory characteristics
Vargas-Bellopérez et al.