Introduction

An emulsifier or emulsifying agent is a compound or substance that acts as a stabilizer for emulsions,

preventing liquids that ordinarily don't mix from separating. The word comes from the Latin word
meaning "to milk," in reference to milk as an emulsion of water and fat. Another word for an emulsifier
is an emulgent. The term emulsifier may also refer to an apparatus that shakes or stirs ingredients to
form an emulsion.

An emulsifier keeps immiscible compounds from separating by increasing the kinetic stability of the

mixture. Surfactants are one class of emulsifiers, which lower surface tension between liquids or
between a solid and liquid. Surfactants keep droplets from getting large enough for components to be
able to separate based on density.

The method of emulsification matters in addition to the nature of the emulsifier. Proper integration of
components extends the emulsion's ability to resist changes. For example, if you are making an
emulsion for cooking, the mixture will maintain its properties longer if you use a blender than if you stir
the ingredients by hand.

Examples

Egg yolks are used as an emulsifier in mayonnaise to keep the oil from separating out. The emulsifying
agent in egg yolks is lecithin.

Mustard contains multiple chemicals in the mucilage around the seed that act together as emulsifiers.

Other examples of emulsifiers include sodium phosphates, sodium stearoyl lactylate, soy lecithin,
Pickering stabilization, and DATEM (diacetyl tartaric acid ester of monoglyceride).

EMULSIFIERS IN FISH NUTRITION AND THEIR IMPLICATIONS FOR PLANT-BASED DIETS

Supplies of fish oil are limited and continued growth in aquaculture production directs that substitutes
must be found that do not compromise fish health and product quality. As a result, several potential
alternative oil sources (plant, animal, algal, etc.) are being evaluated and used in the aquafeed industry.
However, their digestion, absorption, and assimilation are typically lower than fish oil and ultimately
provide lower available energy to fish. An alternative approach, the addition of emulsifier in feed could
enhance lipid digestibility. Emulsifiers have two polar (hydrophilic) and nonpolar (lipophilic) heads
helping water and fat molecules to bind, leading to solubilization of fatty acids and enhanced oil contact
with digestive lipase enzyme. Emulsifiers can be divided into natural and synthetic groups. Bile salts are
one of the common natural emulsifiers, which helps for the digestion and absorption of dietary lipids
and fat-soluble vitamins from the intestine of fish. Bile salts are synthesized by hepatocytes of the liver
through cholesterol conversion, stored in the gallbladder, and secreted to the intestine. These salts
emulsify dietary fat, leading to a larger pancreatic lipase activity. Fat transportation is another role of
emulsifiers, which transport fat from intestinal epithelium to hemolymph and reduce the accumulation
of lipid in the intestine. After digestion, the intestinal bile acids are absorbed along with the nutrients
and returned to the liver via the portal blood. Dietary plant ingredients can affect this circulation. An
increased plant ingredient, such as soybean, reduces gallbladder somatic indices in different species
(Figure 1) and also changes bile salt composition and reduces blood cholesterol levels (Table 1), which
will have negative impacts on fat digestion and absorption.

Dietary supplementation of emulsifiers, e.g. synthetic (Twine 80) and/or natural (bovine salt or lecithin)
can alleviate the negative impact of plant-based diets on the reduction of emulsification capacity of the
bile acid pool. This supplementation also prevents the occurrence of histological abnormalities in the
distal intestine and liver which may be a proper alternative to reduce soybean meal-induced enteritis in
fish. Evidence suggests that fish require emulsifiers when feeding low-digestible oil and/or high-plant
protein diets. Supplementation of emulsifier at the level of 1.5-2% (natural) or 2-4% (synthetic) is
suitable for carnivorous fish to compensate for low fat digestibility caused by plant-based diets.

The importance of digestibility enhancers in current aquafeed formulations by using emulsifying agent

Global aquafeed production is following the exponential expansion of aquaculture and reached close to
40 million tonnes in 2017, with a predicted annual growth rate of 5.5 percent between 2015 and 2025
(Tacon and Metian, 2015). The increasing production volumes resulted for many aquaculture species in
a continuous challenge of the farm gate prices and the profitability of the farm operation. In addition, all
major aquafeed ingredients - including fish meal, fish oil, vegetable proteins and oils, wheat flour, feed
phosphates, additives, vitamins and minerals - have shown significant price fluctuations over the past
years. The increasing replacement of marine ingredients by those of vegetable origin has moderated the
economic pressure on farmers and feed manufacturers and allows for a continuing supply of aquafeed
that can meet the volume required for the developing industry.

Despite economic and volume considerations, feed formulations are not only about price, and the
nutrient supply, absorption and utilization from the new ingredients must be contemplated. The
increasing inclusion of vegetable ingredients requires supplementation with essential nutrients such as
cholesterol, omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), phospholipids, amino acids,
and trace minerals to compensate for the nutritional gaps introduced when replacing marine
ingredients. A recent survey comparing commercial shrimp feeds manufactured in India shows that the
increasing inclusion of plant ingredients between years 2014 and 2016 led to an overall reduction of
16% and 24% in the levels of n-3 LC-PUFA and cholesterol, respectively (van Halteren and Coutteau,
2017). Moreover, plant proteins contain heat-stable anti-nutritional factors, which may induce enteritis
in carnivorous fish species and ultimately have a negative impact on nutrient absorption (Krogdahl et al.,
2003; Romarheim et al., 2006). Given the current trends in aquafeed formulation, it is therefore
important to not only aim for optimising nutritional inputs at the lower cost but also use alternatives
that maximise nutrient absorption and utilization. Digestibility enhancers that optimize the digestive
potential give the formulator flexibility in the use of plant ingredients and formulation of least-cost and
high-quality feeds.

A wide variety of concepts and products are being investigated for improving digestion and feed
utilization in livestock species. The feeding biology, digestive physiology and nutritional requirements of
livestock species differ from those of aquaculture organisms. Therefore, the direct application of
digestibility enhancers in aquaculture requires prior empiric screening with the aim of identifying
effective compounds for the different aquaculture species. Furthermore, a key consideration is that
these compounds must be heat-stable to facilitate the application in the mixer under practical
processing conditions applied in aqua feed manufacturing. The present article describes different
digestibility enhancers and illustrates the potential of their application in aqua feed to reduce cost while
optimising digestive potential and growth performance.

Oils and fats are important sources of essential lipids and energy to the animal. Enhancing lipid
absorption and utilisation is important to maximise the performing efficiency of the plant ingredients
contained in commercial aqua feeds. A first group of digestibility enhancers that target the lipid
components of the feed is emulsifiers. Although lipids are not water soluble, lipid digestion takes place
in a water environment. Emulsifiers are surfactants that lower the tension between water and lipids,
causing the lipids to be broken down into smaller droplets that can be dispersed throughout the water,
and allowing more efficient digestion of triglycerides by lipases. After digestion, emulsifiers associate
with the monoglycerides resulting from digestion and free fatty acids to form micelles that transport
them to the enterocyte surface for absorption.

Phospholipids are well-known emulsifiers, with lecithin likely as the most common form of
phospholipids used in animal nutrition. These are composed of one hydrophilic head group, consisting of
phosphate group and glycerol, and two lipophilic fatty acid tails (Figure 1). The high lipophilic
characteristic of phospholipids makes them excellent emulsifiers for water-in-oil emulsions (ie limited
amount of water is added to a lipid rich environment) such as production of margarines, but weak
emulsifiers for the oil-in-water conditions (ie limited amount of lipids is added to a water rich
environment) such as those of digestion in the gut. Thus, the use of phospholipids in animal feed should
be driven as source to meet animal requirements rather than as strategy to optimise lipid absorption.
Figure 1. Structure of phospholipids and lyso-phospholipids.

The structure of lyso-phospholipids, with only one fatty acid tail, makes them more hydrophilic than
phospholipids, resulting in better capacity to form smaller and increased number of micelles

More efficient emulsification in the gut environment is achieved through the use of lyso-phospholipids,
a derivative of phospholipids following enzymatic hydrolysis. Lyso-phospholipids contain only one fatty
acid tail (Figure 1). This structure makes them more hydrophilic than phospholipids, which translates
into better capacity to disperse lipids, form smaller and increased number of micelles, and consequently
into better absorption. The supplementation of the lyso-phospholipid-based Aqualyso at 0.1% and
during 75 days to feed of carnivorous European seabass with only 16% fish meal and remaining as
vegetable protein resulted in 5% improvement in specific growth rate (SGR %/day) and 4% reduction in
feed conversion ratio (FCR) in relation to control (Figure 2). In rainbow trout feed with 15% fish meal and
30% soybean meal protein, 0.2% inclusion of Aqualysoduring 56 days improved final weight by 5% as
well as SGR and FCR by 4%. Improved performance following Aqualysosupplementation was also
corroborated in herbivorous Nile tilapia, in which 0.1% inclusion in juvenile commercial feed during 75
days resulted in 7% improvement in final weight and 2% improvement in SGR and FCR.
Figure 2. Improvements in growth performance parameters in carnivorous and herbivorous species
following Aqualyso supplementation.

Bile salts are a second type of emulsifier. Unlike phospholipids and lyso-phospholipids, the emulsifying
capacity of bile salts originates from the flat steroidal structure, with the hydrophilic hydroxyl groups on
the concave side and the lipophilic methyl groups on the convex side (Sarkar et al., 2016). The
emulsification properties of bile salts are superior to phospholipids in that a high bile salt:phospholipid
ratio promotes the formation of small size micelles that are more quickly transported to the enterocyte
surface (Cabral and Small, 1989). Besides lipid emulsifiers, bile salts are the major-end metabolites of
cholesterol, and participate in cholesterol homeostasis and in the activation of the pancreatic lipase that
enables fat hydrolysis into monoglycerides (Buchinger et al., 2014).

The inclusion of vegetable proteins, particularly soybean meal, in carnivorous fish species has been
associated to reduced levels or activity of bile salts and consequently to reduced lipid digestibility
(Yamamoto et al., 2007; Romarheim et al., 2008). This has been attributed firstly to impaired bile salt
reabsorption due to intestinal inflammation (Kortner et al., 2013), and secondly to a high loss due to
binding to plant fibers, corroborating the fact that reduced lipid digestibility in fish fed soybean meal is
not always or only associated to enteritis (Refstie et al., 2006). Dietary supplementation of bile salts to
meet the increased requirements associated with the inclusion of vegetable protein can restore or
reinforce the aforementioned functions and therefore promote optimal nutrient digestion and
utilization. This was demonstrated in sea bream and rainbow trout, in which supplementation of bile
salts to soy protein feeds normalized the digestive process as reflected by improvements in lipid and
carbohydrate digestibilities (Takagi et al., 2002; Romarheim et al., 2008). These improvements in
nutrient digestion, particularly of lipids, make bile salts a valuable tool when aiming to reduce the lipid
inclusion levels in the feed formula. In a low temperature diet for trout, lipid reduction from 27% to 23%
and supplementation of bile salts (Lipogest) reduced the cost of feed by 5% without affecting growth
performance and conversion efficiencies.

In conclusion, current trends in aquafeed formulation promote the application of digestibility enhancing
additives such as lyso-phospholipids and bile salts which improve the absorption efficiency of major
nutrients, particularly lipids, and the increasingly limited levels of essential nutrients. This implies the
possibility of extracting more nutritional value of each kilogram of feed, and is particularly important
when aiming to compensate the depressed fat utilisation derived from high plant formulations and to
reduce dietary fat levels without impacting performance.