Stability Aspects of Non-Dairy Milk Alternatives

Abstract

In recent years, plant-based milk products, commonly called as non-dairy milk alternatives have gained high popularity due to concerns associated with bovine milk like lactose intolerance, allergies, hypercholesterolemia, and pesticide and antibiotic residues. Important strategies for manufacture of non-dairy milk alternatives involve disintegration of plant materials in aqueous medium; its homogenization and addition of some additives to attain a consistency and appearance similar to that of bovine milk. Different range of ingredients are added to non-dairy milk alternatives such as oils, emulsifiers, thickeners, antioxidants, minerals etc. The main problem associated with non-dairy milk alternatives is generally linked with its stability. Stability is a crucial factor that governs the sensory properties and overall acceptance of non-dairy milk alternatives. Differences in processing parameters and molecular interaction mechanisms affect the stability of emulsions as well as the stability of non-dairy milk manufactured thereof. Various treatments like thermal treatment, non-thermal processing (ultra high pressure homogenization, pulsed electric field, ultrasonication), addition of emulsifiers are effective in achieving the stability of non-dairy milks. The present chapter aims to summarize the various factors contributing to the physical stability of non-dairy milk alternatives like appearance, consistency, emulsion stability, and the approaches required to maintain it.

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1
Chapter
Stability Aspects of Non-Dairy
Milk Alternatives
JyotikaDhankhar and PreetiKundu
Abstract
In recent years, plant-based milk products, commonly called as non-dairy milk
alternatives have gained high popularity due to concerns associated with bovine
milk like lactose intolerance, allergies, hypercholesterolemia, and pesticide and
antibiotic residues. Important strategies for manufacture of non-dairy milk alterna-
tives involve disintegration of plant materials in aqueous medium; its homogeniza-
tion and addition of some additives to attain a consistency and appearance similar
to that of bovine milk. Different range of ingredients are added to non-dairy milk
alternatives such as oils, emulsifiers, thickeners, antioxidants, minerals etc. The
main problem associated with non-dairy milk alternatives is generally linked
with its stability. Stability is a crucial factor that governs the sensory properties
and overall acceptance of non-dairy milk alternatives. Differences in processing
parameters and molecular interaction mechanisms affect the stability of emulsions
as well as the stability of non-dairy milk manufactured thereof. Various treatments
like thermal treatment, non-thermal processing (ultra high pressure homogeniza-
tion, pulsed electric field, ultrasonication), addition of emulsifiers are effective in
achieving the stability of non-dairy milks. The present chapter aims to summarize
the various factors contributing to the physical stability of non-dairy milk alterna-
tives like appearance, consistency, emulsion stability, and the approaches required
to maintain it.
Keywords: non-dairy milk alternatives, emulsifiers, thickeners, ultra high pressure
homogenization, stability
1. Introduction
Food has served multitude of functions for humans since ages, such as satiat-
ing hunger, quenching the palate with different savory food products, promoting
well-being and socializing on one side of the equation, and providing the basis of
energy production for regulating physiological needs, acting as a source of health
promoting bioactive components, and antioxidants, on other. Among the foods,
animal based products like bovine milk and beef are by far the most commonly
consumed ones in the world. Apart from reasons of health and wellbeing, consum-
ers nowadays are interested in reducing their intake of animal products because of
moral and environmental reasons. Different issues underlying the negative attitude
towards the manufacture of animal based products include environmental pres-
sures from the vast amounts of agricultural produce and water essential for feeding
animals, habitat loss deforestation, animal exploitation, species extinction, and pol-
lution in production and transportation of the food until it reaches the consumer.

Milk Substitutes - Selected Aspects
2
Since in recent years the animal based diet is being negatively associated with the
individual’s health and the environment, people have started looking for other food
options [1]. Consequently, the plant based diet has become a favorite among people
because of its potential to promote health, to improve food security, and to decrease
pollution, land use, and water use [2].
Because of the increase in the global urban population, and with the consumers
having more purchasing power and health awareness nowadays, the demand for
healthier, tastier, and newer food products has risen tremendously. Furthermore,
research for various innovative and novel food product developments in the last
decade has been focused on meeting the emerging needs and adapting to existing
market demands by providing newer food choices and alternatives. Therefore, the
plant based diets like non-dairy milk alternatives, in particular, seem to have experi-
enced a surge in the market. Besides, there is increasing negative perception related to
the consumption of bovine milk among consumers as it has been linked adversely to
many diseases such as bovine milk allergy, lactose intolerance, anemia, and coronary
heart diseases for the past many years [3–5] and also due to issues that have raised
concern in recent years, like the presence of toxic chemicals, antibiotics, contami-
nants, and greenhouse gas emissions. Nondairy milk alternatives possess health
beneficial components, including antioxidant, antimicrobial, dietary fibers, unsatu-
rated fatty acids; and hence, are desirable among consumers [6–8]. Nonetheless, the
market for non-dairy milk alternatives is still emerging and currently, the range of
products available in the market include hazelnut, peanut, sesame, soy, almond, oat,
rice, hemp, and walnut milk; issues regarding the stability and nutritional value is
still a concern among consumers. For successful commercialization of non-dairy milk
alternatives, processors are often interested in the technological interventions and
ingredients that can help maintain the physical stability of the final product. Physical
stability refers to the maintenance of inherent attributes of suspension in relation to
its viscosity, appearance, consistency, color, and resistance to destabilization mecha-
nisms like sedimentation, phase separation, flocculation, creaming, etc. The general
manufacturing process involves soaking the raw material (nut, legume, cereals,
pseudocereal) in water, disintegrating moist material, separating oil bodies, adding
different additives, heating for killing the harmful microorganisms, homogenization,
and aseptic packaging [9]. Technological interventions are required to manufacture
milk substitutes equivalent to bovine milk in their appearance, flavor, stability, and
nutritional components. Most of these milks are unstable during manufacturing and
storage; they tend to undergo phase separation and spoilage on long term storage.
For these reasons, various methods have been employed to achieve stability in these
non-dairy milks, for instance, by incorporation of different types of additives, such
as gums, thickeners, emulsifiers, and by application of new technologies, like ultra-
high-pressure homogenization, ultrasound, and pulsed electric fields. Therefore,
while formulating non-dairy milk alternatives, it is necessary to endeavor towards
utilizing the beneficial properties of plant materials and employing appropriate tech-
nologies for manufacturing non-dairy milks such that they are stable, display func-
tional characteristics and sensory attributes similar to those of bovine milk. The aim
of this chapter is to discuss the processing steps, mechanisms underlying the physical
instability and to explore possible solutions with regard to use of different additives
and advanced technological interventions in manufacture of non-dairy milks.
2. Need for stability of non-dairy milk alternatives
Bovine milk is nature’s most complete food [10] with different components
present in heterogeneous mixture like carbohydrates, whey proteins and minerals

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in solution; fat globules in emulsion while casein micelles and some minerals are
distributed in colloidal phase, giving the bovine milk its typical composition and
structure [11]. Being a rich source of nutrients, bovine milk is a perishable food item
and is often subjected to heat treatments, like pasteurization to extend shelf life for
a week on refrigeration; UHT for shelf life extension to several months at ambient
temperature [12]. In general, different processing operations, like heat treatment
and homogenization are greatly influenced by the structural design of bovine milk
components conferring it suitability for use in different food systems [13]. However,
in case of the plant-based milk alternatives, sales trend suggest that the customers
are hesitant to buy them because they display undesirable behavior when served hot
or on blending with the hot drinks.
Therefore, the beneficial qualities of the bovine milk must be closely reproduced
by plant based milks, if they are to be perceived better than or equal to them.
During the formulation of plant-based milk substitutes, it is essential to adopt
effective technologies and suitable ingredients to achieve the stability to overcome
the problems of unacceptable flavor and phase separation on storage, commonly
associated with the beverages. Different novel technologies that have been applied
for achieving stabilization involve reduction in particle size, decrease in viscosity,
and decrease in microbial count [14–19].
It has been demonstrated that size of dispersed phase particles in plant-based
milks is one of the important factors governing their stability [15, 20, 21]. Plant-
based milks are colloidal dispersions consisting of wide range of components such
as fat globules, ground raw material, proteins and carbohydrates etc. They often
contribute to unstable product on storage as they tend to exhibit phenomena like
creaming, sedimentation and phase separation. Besides, non-dairy milks are often
associated with sandy, gritty or chalky mouthfeel and tend to develop off flavors
during storage [22, 23]. Also, during formulation of non-dairy milk substitutes,
bovine milk fat globule is an ideal candidate that needs to be simulated due to its
significant contribution to the creaminess, texture and flavor of dairy products. To
develop non-dairy milk alternatives, fat phase is incorporated either through addi-
tion of oil bodies [24] or fabrication of fat globules from plant sources [25].
It is essential to take different aspects in account, such as kind of raw material,
shelf stability, processing operations and various electrostatic interactions underly-
ing phase destabilization (creaming, flocculation, sedimentation, coalescence)
while manufacturing non-dairy milks. With regard to stability of non-dairy milk
alternatives, one fundamental attribute that is relevant to most of the products is
their colloidal nature since other features like composition, and structure often vary
markedly among different brands. Therefore, different characteristics that need to
be monitored accurately in non-dairy milks, include properties of colloidal par-
ticles such as their size, charge density, surface charge, and surface properties, the
nature of the continuous aqueous phase (the pH, ionic concentration, components,
density, and viscosity), and the extent of exposure to external environment during
its shelf life (storage temperature and time). Plant based milks not only undergo
objectionable changes in physicochemical properties but also show signs of micro-
bial spoilage on long term storage. Some of the necessary ingredients, processing
techniques, and phenomena governing the physical stability of plant-based milks
during manufacture and storage have been discussed below.
3. Processing steps for manufacturing non-dairy milk alternatives
During the manufacture of non-dairy milk alternatives, they are often subjected
to various preprocessing treatments like dehulling, soaking, sprouting, blanching

Milk Substitutes - Selected Aspects
4
etc., to assist in subsequent processing. In general, the processing of milk from
plants involves two main methods, namely, wet and dry. Otherwise, product is
formulated by reconstitution using protein isolates or concentrates, water and
other ingredients like oils, sugars, salts and stabilizers [26]. In the wet process,
plant based raw material is soaked and ground with the water into a slurry, while
in the dry method, the plant based material is ground into flour and then extracted
with water. Such material is then subjected to filtration to remove insoluble or
coarse particles to obtain aqueous phase. Afterwards, the processing steps followed
include the addition of ingredients like oil, sugar, salts, colors, flavors, and stabiliz-
ers; homogenization and thermal processing treatments to yield non-dairy milk
alternatives with desirable attributes.
3.1 Preliminary processing treatments
.. Dehulling
Dehulling operation involves the mechanical breaking of thick and hard seed coats
of plant based raw materials before soaking to facilitate hydration. The strength of
binding of the hull to endosperm governs the time required for dehulling procedure.
Since the hull has a hydrophobic nature due to its association with hemicellulose and
pentosans, it tends to lower down the hydration capacity of plant material. The poly-
saccharides present in the hull often lead to off-flavor, and foaming during processing,
hence, their removal improves the processing operation and organoleptic properties
of product. Also, microorganisms and enzymic activity associated with hulls reduces
on dehulling. The traditional method includes initially exposing the raw material
to the sun for drying and then dehulling with mortar and pestle. Alternatively, they
are dehulled using the mills, and may also be milled using splitting machine, which
employs both splitting and dehulling simultaneously. The milk prepared from dehu-
lled raw material allows for production of a shelf stable and appealing final product.
.. Roasting
Roasting is a thermal process encompassing dehydration of raw material [27] for
its improved flavor, aroma, and milling properties. Decrease in protein, starch con-
tent and improved extraction yield of roasted pulses and grains have been reported
by many authors [28, 29]. Studies have shown that roasting leads to improved
protein digestibility, and reduction in antinutritional compounds found in raw
pulses and nuts. The decrease in protein content has been ascribed to the partial loss
of amino acids, as well as of some nitrogenous compounds, and the reduction in
starch to the solubilization of starch during the thermal process. Also, roasting has
been shown to increase the water absorption capacity and water absorption index.
An increase in WAC and WAI is related to the denaturation of proteins and starch
gelatinization, which contribute to enhanced water imbibition [30]. Therefore, the
flours with higher WAC are likely to result in the more viscous non-dairy alterna-
tive compared to untreated ones. Thermal processing during roasting results in
partial disruption of the raw material [31, 32], thereby facilitating efficient particle
size reduction required for stable suspension of non-dairy milk alternatives. For
manufacturing milk from nuts and seeds, which contain high levels of unsaturated
fatty acids, roasting should be carried out in controlled conditions of time and
temperature to improve their nutritional properties and for prevention of off flavor
development due to oxidation of unsaturated fatty acids. Inactivation of lipoxygen-
ase during the process improves the flavor of non-dairy alternatives like soy milk,
peanut milk, melon milk, sesame milk [33–37]. Roasted plant material becomes

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drier and brittle, and the non-dairy alternatives obtained from them are likely
less-creamy [36]. In the study for manufacture of sesame milk, it was studied that
the roasting process decreased acidity, total solids content and improved sensory
profile by decreasing bitterness and a chalky taste associated with the milk [37]. The
product obtained upon roasting has improved nutritional and sensory properties.
.. Sprouting
Sprouting refers to the soaking of seeds in water for specified time (1–14hours)
depending on the kind (variety, size, shape) of food grains in order to hydrate
them for breaking their dormancy. The soaked grains are subsequently drained and
rinsed at regular intervals to enable sprouting. Sprouting results in the initiation
of series of metabolic changes in seeds (legumes, cereals, nuts & oilseeds) that
improves the nutritional quality by inactivating the anti-nutritional factors such
as trypsin inhibitor and phytic acid [38]. The improvement in nutritional value
occurs due to enhanced activities of hydrolytic enzymes, which cause the conver-
sion of stored chemical compounds, such as protein, starch and lipids into simple
compounds; thereby, increasing the levels of total proteins, fat, certain essential
amino acids, total sugars, B-group vitamins and decreasing the levels of starch.
Therefore, the sprouting of raw material assists in the development of non-dairy
milk alternatives, which are generally prepared using the heat treatment to decrease
anti nutrient factors. Because sprouting is a natural biochemical process involving
enzymatic activity, the treatment yields the improved quality of final product in
terms of the nutrient and sensory value. Such a treatment decreases the intensity
of heat treatment required for the manufacture of the product. Sprouting ensues
improved protein solubility and reduced fat content for raw materials, which
decreases the viscosity of non-dairy milk alternatives [38, 39]. Also, improvement
in sensory properties takes place due to absence of beany flavor.
.. Blanching
Blanching with hot water is employed to inactivate enzymes like lipoxygenase
and trypsin inhibitors for improvement of the flavor and nutritional value of the
non-dairy milk alternatives [40]. Such a treatment has been reported to be effec-
tive in diminishing the beany, grassy, bitter, and rancid flavor; it also prevents
suspension instability and chalkiness in non-dairy milks prepared from peanuts,
soybean, almonds etc. [41–45]. Blanching with hot water (85–100°C for 2–5min)
is commonly used for skin removal of raw materials and overcoming off flavors in
non-dairy milk alternatives. Like roasting, blanching inactivates enzymes, reduces
possible microbial contamination, and aids in deskinning in processing by wet or
dry methods [46]. Pressure blanching (at 121°C, 15psi for 3min) has been found to
be effective for developing peanut milk with desirable sensory and physicochemi-
cal properties [47]. The treatment yields the milk with pleasing sensory attributes
because blanching treatment for suitable time decreases the total solids and nutty
flavor associated with peanut milk. The treated milk has improved consistency as
well as decreased soaking time.
3.2 Wet processing
.. Soaking
The process for manufacturing of non-dairy milk alternatives involves the
soaking of raw material in the proper volume of water contained in large stainless

Milk Substitutes - Selected Aspects
6
steel containers. Soaking is done to hydrate the raw material (cereals, legumes, nuts,
or seeds) for grinding and further processing. Time required for soaking depends
on the nature of raw material and temperature of the soaking water. At an ambi-
ent temperature, soaking requires longer time, and souring may take place due to
bacterial growth, whereas if the temperature is raised up to 50–80°C, soaking time
is decreased, and hydration is accelerated. It has been demonstrated that during
the soaking of lentils at different temperatures (20, 50, and 80°C), rate of hydra-
tion at 50 and 80°C was four to six fold higher than at 20°C [48]. Softening due to
soaking at higher temperatures could be related to the heat-induced modification in
biomolecules, including starch, pectin, and protein, and the moisture for making
the biomolecules susceptible to the changes. Different processes for preparation
of non-dairy milks like peanut, soy, almond milk include soaking the raw material
for 12 to 18h before grinding it either in the mixer grinder or in colloidal mill [45].
Soaking facilitates the inactivation of enzyme inhibitors, improves digestibility and
bioavailability of nutrients [49]. In case of pulses and grains, soaking step reduces
the polyphenols and eliminates the alkaloids (e.g., in lupin) present in some of
them; decreases the cooking time; improves the protein bioavailability and assists in
peeling or dehulling [50, 51]. Soaking in acidic or basic solution is done to facilitate
peeling of walnuts, almonds, tiger nuts, Brazilian nuts etc. Studies have shown
that basic solution (1–2% NaOH) is suitable for peeling of walnuts and Brazil nuts
[52–53] while citric acid is effective for peeling tiger nuts [54].
.. Wet milling or grinding
The procedure involves the grinding or milling of the plant material with the
use of water for the split opening of the exterior hull. Wet grinding consists of
grinding of fresh raw materials with the water to result in a suspension. The wet
grinding method tends to produce finer particle size of the ground material [55]
that results in more stability of non-dairy milk alternatives, and therefore, is more
commonly used for their manufacture. In general, a colloid mill is used for reduc-
ing the particle size of raw material in suspension. Initially, the coarse grinding of
raw material is done, which is followed by fine grinding. During the wet milling
with the colloid mill, the rotor generates a substantial amount of stress by the
rotation of the rotary stirrer, which can effectively accomplish the creation of sub-
micron particles. In addition to disintegration, the colloid milling performs broad
spectrum of functions like mixing, blending, and homogenizing effects [56]. In
the manufacturing of the non-dairy milk alternative, this technique is mostly used
for homogenization and emulsification [57]. The optimization of colloidal milling
process parameters improves the physical stability of non-dairy milk alternatives
by efficiently reducing the size of dispersed particles [58]. Different studies have
shown that the amount of water added for wet milling depends on the kind of raw
material, for instance, almond milk (1:9; almond& water), Sesame (1:5; sesame
& water), Peanut (1:9; Peanut: water), soybean (1:5; soy: water) [15, 36, 37, 59].
Wet milling contributes the formulation of stable product where different factors
like rotor speed, temperature, ratio of raw material and water can be fine-tuned to
achieve any kind of non-dairy milk alternative.
3.3 Dry processing
The dry process comprises drying the raw materials and milling them into
flours. For improving the efficiency of dry grinding, the raw material should be
dried to minimum water content. The flour may be subsequently treated to yield

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different fractions: the protein, the starch and fiber. The protein concentrate
or isolate, afterwards, is often utilized in formulation of non-dairy beverage.
Therefore, dry processing mostly leads to development of product with higher
protein contents.
.. Dry milling or grinding
The dry milling is mostly employed to reduce the particle size of the dried
raw materials into their respective powder forms. The ground material is then
mixed with water to form paste. However, during the manufacturing of the
non-dairy alternative from pastes, solids tend to settle out down in the container,
thereby resulting in the incomplete transfer of the content to the homogenizer
and its wastage as well. For ensuring the efficient fuctioning of the dry grinding
process, the important factors to be considered are the particle stiffness and feed
size. Although the dry grinding decreases water wastage and energy consump-
tion, yields a product with higher quality of protein, carbohydrates, fat, and
minerals, it is less popular due to handling problems, like dust and wastage of
raw material.
3.4 Extraction
For manufacturing the non-dairy milk alternative, the raw material, once
it has been subjected to preliminary processing treatments, is extracted with
water. The extraction efficiency can be improved by variation of pH or enzymatic
treatment.
.. Extraction by variation of pH
The pH during extraction dictates the efficiency of protein extraction and sta-
bility of emulsion in non-dairy milk. Globulins comprise a major fraction of plant
proteins, while albumins represent a minor fraction [60–62]. The pI for globulins is
near pH4.5, whereas the pI of albumins is around pH 6. The pI for different plant
proteins lies between these values. Different studies have also demonstrated that
plant proteins like pea, lentil, chickpea, soy etc. have a low net charge around pH5
[63]. The plant proteins are mostly stable to pH changes at all pH values except at
pH 5, which is around the pI where the droplets carry no charge and tend to display
phase destabilization phenomena like aggregation and flocculation. During extrac-
tion, proteins should a have a high net charge at pH values well above or below their
isoelectric point, which solubilizes them to increase the yield. Extraction in alkaline
pH exhibits improved protein extraction yield, which may then be followed by neu-
tralization step. For achieving the higher yield of the process, the efficiency of this
step may be improved by alkalization of the medium using bicarbonate or NaOH.
.. Extraction using enzymatic treatment
Enzymatic treatment for hydrolysis of proteins and polysaccharides is mostly
employed to improve the extraction yields. Disruption of plant cell wall components
like cellulose, hemicelluloses, and pectin is facilitated by enzymes to improve the
yield. The efficiency of protein and oil extraction is closely related with cell wall
disruption of plant based material [64]. Studies have shown that cell wall degrad-
ing enzymes with pectinolytic activity like polygalacturonase, pectate lyaese, or
pectin methyl esterase enhance the extractability of protein, fat, and antioxidant

Milk Substitutes - Selected Aspects
8
activity [65–67]. Also, upon application of cell wall degrading enzymes (cellulase,
hemicellulase, pectinase) after homogenization step helps reduce the particle size,
thereby facilitating suspension stability [68]. Because of the reduced particle size of
suspended material, the enzyme treated non-dairy milks exhibit improved physical
stability and flavor. Rosenthal etal. [68] reported that enzymatic treatment (1.2%
of Celluclast) decreased the tendency of soymilk to undergo sedimentation on
storage and improved sensorial attributes in terms of improved viscosity and lack of
chalkiness. Proteolytic enzymes tend to improve the extraction yield and suspension
stability [69]. A high solubility is required for the proteins because it governs their
functional properties, for instance, emulsification, which subsequently affects the
colloidal stability of the emulsion. The extraction of protein also increases due to
improved solubility of proteins. Other enzymatic treatments involving the use of
carbohydrate degrading enzymes like amyloglucosidase, amylases etc. have been
demonstrated to improve the carbohydrate recovery and stability of non-dairy
milk alternatives. Depending upon the plant based material containing appreciable
amount of starch, for instance, in case of cereals & pseudocereals, liquefaction with
α-and β-amylases is done for starch hydrolysis [70–72]. Upon heating, starch gelati-
nizes to set as thick gel during heating, and hence enzymatic treatment is required
to maintain the non-dairy milk in the liquid state. The liquefaction treatment
increases the yield due to hydrolysis of starch into maltodextrin, thereby improving
the viscosity for the non-dairy milk alternative. Since it facilitates the filtration, the
enzyme treatment is often employed during or before filtration; however, it might
also be used after filtration, subject to the conditions. Studies have shown that starch
liquefaction using amylases generally improves the viscosity and overall acceptabil-
ity in non-dairy milk alternatives like oat milk, quinoa milk, rice milk, [72–74].
3.5 Filtration
Following the extraction step, removal of okara (the water-insoluble portion)
from the slurry is done to obtain aqueous portion for manufacturing non-dairy
milk alternative. The separation step is achieved by employing either batch process
using filter cloth or continuous process like centrifugation [75, 76]. In general,
two stage centrifugation is carried out to improve the efficiency of separation. In
two stage clarification, separation of okara is carried out in first stage while fine
particles are separated in second stage. Efficient filtration enables the retention
of fine particles in the aqueous phase which assists in achieving the suspension
stability. Different studies have shown that filtration treatment through a decanter
or continuous filtration system (20–80μm) during the manufacturing process
of non-dairy milks improves the physical stability of milk because of removal of
suspended particles [68, 77]. These days membrane separation is becoming popu-
lar as it allows for efficient separation of aqueous portion from okara. In case of
manufacturing milk from fat rich raw material, the surplus fat is separated using a
separator as is done in dairy processing with cream separator.
3.6 Addition of ingredients
Once aqueous phase or base material is obtained upon extraction and filtration,
other ingredients are blended in the aqueous phase in optimum levels for success-
ful manufacturing of non-dairy milk alternatives. These ingredients include fat,
vitamins, sugar, flavorings, salt, oils and stabilizers etc. Since physical poses a chal-
lenge for the successful development of any non-dairy milk alternative, different

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range of additives (emulsifiers and stabilizers) have been explored for their use in
the milks. Various emulsifying agents such as alginates, gelatin, xanthan gum, gum
Arabic, locust bean gum, and gellan gum in a range of 0.5 to 1% by weight dem-
onstrate improved emulsion stability. The destabilization due to settling of solid
particles in the emulsion may be overcome by addition of alkalizing agents, such
as disodium phosphate or sodium bicarbonate. Maghsoudlou etal. [16] achieved
stability of almond milk by using lecithin, modified starch and agar at 0.09%,
1.31% and 0.15% levels respectively. Nor (2012) suggested that addition of lecithin
(0.03% w/w) at the time of the milling during manufacture of almond milk was
beneficial for its stability. Hinds etal. [78] reported good results with the use of
0.02–0.04% carrageenan as stabilizer in peanut milk. Bernat etal. [20] established
that addition of 0.05g/100mL xanthan gum before the heat processing was suit-
able for developing hazelnut milk substitute as it causes thickening of the hazelnut
milk substitute and enhances the colloidal stability of the final product. Processing
operations should be performed carefully, since non-dairy milk alternatives are
fortified with minerals and vitamins which may compromise the stability of
emulsion. This is because vitamins are known to exhibit instability in relation to
environmental conditions like high temperature, light and exposure to oxygen.
In addition, mineral fortification might result in destabilization of emulsion;
therefore, their fortification is accompanied with the addition of chelators like
citric and EDTA. Based on the dispersibility and solubility of mineral sources, the
salts that are commonly used for the mineral fortification include ferric gluconate,
ferric ammonium citrate and ferric pyrophosphate as iron sources and calcium
citrate, tricalcium phosphate and calcium carbonate as calcium sources [79, 80].
3.7 Homogenization
Homogenization is employed for size reduction of the dispersed phase com-
ponents in the range of 0.5–30μm by application of shear forces. The particles
of the dispersed phase like protein, starch, fiber, and other cellular materials
tend to sediment at the bottom when allowed to stand for some time; however,
with the contribution of size reduction due to homogenization and addition of
emulsifying agents or hydrocolloids, the stabilization of suspension is achieved
during manufacturing of non-dairy milk alternatives. For carrying out homog-
enization, a pressure range of 20–60MPa has been employed to improve the
suspension stability during manufacture of non-dairy milk alternatives like rice,
hemp, coconut milk [81–83]. The process assists in subdivision of fat globules to
prevent phase separation and facilitates development of creamier and homog-
enized product.
3.8 Heat treatment
High temperature treatments like pasteurization, sterilization or UHT are
employed to increase the shelf life of non-dairy milks by destruction of microor-
ganisms. Several studies have reported application of sterilization treatments at
121°C for 15–30min in various non- dairy milks like almond, soy and peanut milks
[20, 81, 84, 85]. Also, UHT treatment in range of 134–140°C for 2 to 20seconds has
been applied in different non-dairy milks like peanut, coconut and almond milk
[69, 86]. However, high temperature treatments have been reported to destabilize
non-dairy milk alternative by resulting in coagulation of proteins. This is because
proteins at high temperatures unfold to expose nonpolar amino acid residues,

Milk Substitutes - Selected Aspects
10
which participate in protein–protein interactions and consequently, exhibit aggre-
gation, sedimentation, or gelling phenomena. Homogenization treatment after heat
processing improves suspension stability by disruption of aggregates and reduction
of particle size distribution [87]. The gelling and thickening of non-dairy milks
due to presence of starch is addressed by enzymatic treatment for hydrolyzing the
carbohydrate. Apart from enhancing physical stability, these heat treatments cause
simultaneous destruction of pathogenic microbes in plant based milk alternatives
resulting in increased storage stability of these beverages. Maria etal. [88] reported
that the pasteurization treatment improved the quality characteristics of almond
milk. Likewise, Khodke etal. [89] evaluated the effect of sterilization on shelf life
of soymilk. It was observed that sterilized samples were acceptable up to 90days
at ambient storage while at refrigerated storage, the shelf life of milk samples
increased up to 170days. In another study, the effect of ultra-high temperature
treatment on quality attributes of soymilk was investigated. It was concluded that
single step UHT process (143°C/60s) can result into a commercially sterile soymilk
with thiamin retention up to 93%, reduced trypsin inhibitor activity and improved
acceptable sensory properties [90].
3.9 Aseptic packaging
Aseptic packaging of non-dairy milk alternative into sterile packaging material
is done to increase the shelf life of the product.
4. Influence of ingredients on the stability of non-dairy milk
alternatives
4.1 Important ingredients
.. Fat phase
In formulation of nondairy milks, fats are standardized in products either as oil
bodies obtained from plants or are fabricated synthetically through homogeniza-
tion. Oil bodies consist of a fatty acid core made up of triacylglycerol and a sur-
rounding monolayer of phospholipids and unique proteins (oleosins), thus which
imparts a structure composition similar to that of milk fat globule [91]. Extraction
of oil bodies from plant seeds is generally achieved by employing physical pro-
cesses, like soaking and crushing to enable their separation from adjacent tissues
[9]. Oleosins play important role in stabilization of oil bodies by preventing their
coalescence [92], preventing their hydrolysis by phospholipases [20] and by bal-
ancing of PUFA to MUFA ratio [93]. Even though plant based milks are similar to
bovine milk, they may exhibit a distinct flavor, perceptible as nutty or beany, and
may not be as desirable compared to flavor of milk [24] which is mild and unique
owing to its typical aroma and taste profile [94].
Owing to differences between the dispersed and continuous phases in colloidal
dispersions, there is a net movement of particles between two phases under the
influence of gravitational force; creaming occurs if density of particles in dispersed
phase is lower compared to dispersion medium whereas sedimentation is evident, if
the case is otherwise. Both these phenomena tend to destabilize a colloidal disper-
sion. With respect to non-dairy milks, oil bodies tend to exhibit upward movement,
while raw material fractions and being heavier, tend to settle down at bottom
resulting in sedimentation, and is usually overcome by homogenization. Besides,

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simulated fat may also be stabilized by use of emulsifiers and homogenization,
thereby imparting to non-dairy milk alternatives the characteristics similar to those
of bovine milk in terms of consistency, appearance, flavor, and mouthfeel [95].
Fat phase in plant based milks is derived from different oil sources like coconut,
palm, sesame, flaxseed, sunflower, olive, and soybean which contribute to different
attributes like solid fat index, melting/crystallization pattern, viscosity, sensory
and physicochemical properties. These features have important implications on
processing of non-dairy milk alternatives such as presence of molten state of fat
prior to homogenization and subsequently size of oil droplets created. However,
presence of unsaturated fatty acids in lipid phase of these milks renders them more
prone to lipid oxidation and rancidity. In case the ratio of unsaturated to saturated
fatty acids is high, it contributes positively to human health. Numerous studies over
the years have associated the consumption of plant-based oils with beneficial health
effects, such as anticarcinogenic, anti-inflammatory, anti-dyslipidemia, antioxi-
dant and in particular, improved cardiac health status has been attributed to intake
of unsaturated fatty acids [96, 97].
Because of the density difference that exists between the dispersed phase and
continuous phase, gravitational separation is a phenomenon commonly observed
in non-dairy milk alternatives. In order to overcome phase separation, the den-
sity difference may be diminished either by incorporating in the milk alterna-
tives the fat with the higher solid fat index or by adding some weighting agents,
surfactants, and biopolymers that can hold onto the oil bodies by completely
surrounding them. Creaming is controlled either by formation of tenacious
films by proteins on oil droplets or by increase of viscosity of the medium, for
instance by addition of thickening agents like hydrocolloids and polysaccharides
to the dispersion medium. It is because when there is incomplete coverage of the
oil body, partial coalescence may take place, and aggregation occurs in the fat
bodies in such cases. In general, the difference between the density of the aque-
ous and fat phase may be adjusted by the use of weighting agent like brominated
vegetable oil. However, brominated oil is not commonly used in food emulsions
since it has been shown to negatively affect the fat metabolism in rats [98].
Addition of brominated vegetable oil to regular vegetable oil at 25wt% level
diminishes the density difference between oil phase and aqueous phase [99].
Therefore, in order to achieve stability in milk alternatives, it is essential that
lipid bodies may be designed either using fats with proper solid proportion to
increase the density of dispersed phase or using suitable biopolymers for ensur-
ing efficient coverage.
Flocculation is a phenomenon that involves the weak association of oil droplets
due to net attractive forces resulting in formation of flocks. The characteristics of
the flocks vary with the extent of the net force of attraction between the droplets
and the oil volume fraction. In the cases when the net attractive forces are not
strong, weak flocculation occurs, while large aggregates formation takes place due
to strong attractive forces in the non-dairy milk alternatives. Flocculation of oil
droplets leading to instability of the milk substitutes is governed by non-covalent
interactions which may be either attractive (van der Waals forces) or repulsive
(electrostatic forces and steric forces) and can be manipulated by using appropri-
ate surfactant or biopolymer. The additive should present the properties capable
of generating stronger repulsive forces compared to attractive forces to overcome
aggregation. Surfactants, cationic or anionic in nature, upon formation of films,
generate electrostatic forces, which stabilize the oil droplets against aggregation
due to net repulsive forces. However, proteins are quite effective in stabilization
against aggregation owing to strong steric repulsive forces associated with them.

Milk Substitutes - Selected Aspects
12
Adsorption of fat droplets by proteins, causes overlap of the outer portion, which
entails an osmotic pressure gradient; thereby, generating the repulsive forces
which prevent droplet aggregation. This leads to decrease in entropy and overall
stabilization of non-dairy milk.
.. Emulsion stabilizer
Since non-dairy milk alternatives are typically oil in water emulsions present
in complex multi-component systems entailing fats, proteins and polysaccharides,
additives, water, sugars, flavors, other small molecular-weight compounds, and are
inherently unstable exhibiting phenomena like aggregation, creaming, coalescence,
sedimentation. Therefore, it is essential to select relevant emulsion stabilizer (surfac-
tants, emulsifiers and hydrocolloids) for improvising the stability of milk substitutes.
.. Different types of emulsifiers
Emulsifiers are usually surface active molecules that act by adsorbing to
the surfaces of the droplets of dispersed phase by creating a protective coating
around them to prevent their aggregation. They may be categorized in different
forms like, low molecular weight compounds: synthetic (monoglycerides, poly-
glycerol esters) or natural (phospholipids) and high molecular weight biopoly-
mers (proteins and polysaccharides) [100–102]. As to the stability of emulsions
imparted by emulsifiers, it is mainly related to formation of viscoelastic films
around dispersed droplets. Several studies have suggested that the main cause of
stabilization of emulsions is related to the capacity of emulsifiers to efficiently
adsorb on dispersed droplets, size of the droplets, concentration of emulsifier,
and generation of repulsive forces as well as considerable reduction of surface
tension [95, 103, 104].
.. Low molecular weight surfactants
Food industry has always shown interest in use of suitable emulsifiers in differ-
ent formulations as various features of food are influenced like stability, mouthfeel,
color, flavor, appearance, texture and shelf life of food. Low molecular weight
surfactants (phospholipids, monoacylglycerol) are more efficient than proteins
in reducing the interfacial tension between two phases of an emulsion because of
their property of quick diffusion and adsorption to interface [105–114]. Proteins
on account of being bulky are slow to diffuse to interface and hence, exhibit lower
surface activity [105]. This might be attributed to the complex structure of a
protein consisting of both hydrophobic and hydrophilic groups present variably
throughout its primary structure, and as separate patches in tertiary structures with
no clearly defined head and tail region, which, are essentially distinct in case of
small surfactants. Moreover, due to the absence of conformational constraints for
rearrangement at the interface, low molecular weight surfactants, at sufficiently
high concentrations, are more successful than proteins prevent adsorption to oil
droplets. In the case of emulsions, when the protein to surfactant ratio is low,
protein displacement into the continuous phase takes place due to the surfactant
molecule, based on the orogenic mechanism [102]. The mechanism suggests that
the protein molecules are unable to pack completely, and adsorb homogeneously on
the interface because of steric hindrance, thereby creating a void space. The void
spaces are primarily occupied by the surfactant domains, which enlarge gradually
creating pressures, that compress the nearby protein film, and finally resulting in its
desorption in the continuous phase [113].

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.. Proteins
Proteins adsorb to oil droplets by undergoing partial denaturation to posi-
tion themselves such that buried hydrophobic residues are exposed to the oil
phase while hydrophilic residues align towards the aqueous phase [13]. On
diffusing to the interface, proteins form tenacious viscoelastic films which are
not apparent with the surfactants. The films are able to withstand mechani-
cal stress and impart electrostatic as well as steric stabilization corresponding
to type of protein and solvent conditions [115]. In these emulsions, stabil-
ity may also be attributed to presence of “loops and trains” in protein chain
conformation[116–118].
Among the natural class of emulsifiers, proteins represent very interesting emul-
sifiers due to their film forming ability and amphipathic nature [119]. Generally,
animal proteins have been popular in food industry due to their excellent emulsify-
ing abilities. These include bovine milk and egg proteins such as casein, whey pro-
tein isolate, bovine serum albumin, ovalbumin, ovotransferrin [120–128]. However,
during recent years, plant proteins have experienced increasing popularity among
manufacturers because of their association with several beneficial properties such
as stability, sustainability non-allergenicity, non-toxicity, low-cost, biodegrad-
ability, functional properties, and consumer acceptance due to the clean label status
ascribed to them [129, 130]. Different plant proteins that have gained acceptance
as emulsifier in various emulsion based food systems include soy proteins, chick-
pea, lentil, cowpea, pea proteins wheat gluten, rice glutelin and flaxseed protein
[131–134]. In order to stabilize emulsions successfully, it is necessary that emulsi-
fiers should not only prevent droplet aggregation but also be stable to exterior
stresses like temperature, pH, salt concentrations, sugars, etc. Biopolymers such as
proteins and polysaccharides vary in stability with respect to external conditions.
Plant proteins (pea, legume, faba bean) lack stability at pH close to their pI, high
temperature and high salt concentrations [135], whereas polysaccharides exhibit
stability under similar conditions [136, 137].
Plant proteins are generally globular, like soy, pea, chickpea and cereal protein
which undergo entropy changes on adsorption at interface through structural
rearrangement in secondary and tertiary changes [138]. Likewise, in bovine milk,
the whey proteins are globular in nature. β-lactoglobulin usually has much the
unordered structure and α-lactalbumin helical structure. In contrast, the com-
plex globular proteins from plant sources have ordered structure. For instance,
the legume proteins such as glycinin and legumin have well-ordered and greatly
conserved structure due to their rigid quaternary conformation. The quaternary
structure undergoes conformational deformation at tertiary and secondary
configuration on getting adsorbed at the interface. Proteins that have inherently
disordered structures show better surface activity compared with ordered proteins.
Unstructured proteins like casein, which have open random coil structure, exhibits
conformational rearrangement as an emulsifier causing fast changes compared to
globular proteins. Studies have shown that the competitive adsorption of proteins
takes place at the oil–water interface in non-dairy milks, and among the mixture
of proteins, some proteins adsorb more effectively compared to others based on
their structure and the partitioning of hydrophobic and hydrophilic residues.
Moreover, as plant proteins are globular, the exposed hydrophobic groups tend to
adsorb to nonpolar groups of oil droplets in non-dairy alternatives, ensuing strong
and long-range hydrophobic attractive forces, which overcome the repulsive forces,
so that the net effect is particle aggregation. Therefore, the important aspect for
control, in the viewpoint of the manufacturers to ensure the stability, is hydropho-
bicity, which is the inherent characteristic of the globular proteins, and besides,

Milk Substitutes - Selected Aspects
14
it becomes more pronounced due to thermal or surface denaturation. In order
to prevent hydrophobic flocculation, it is necessary to select suitable proteins,
which are less hydrophobic, and to avoid the processing procedures that encourage
protein denaturation.
Therefore, to achieve stability in plant based milks, certain protein modification
strategies may be applied. As discussed above, globular proteins are susceptible to
denaturation, their surface activity and solubility may be altered during processing
of non-dairy alternatives [139]. Physical, chemical and enzymatic modifications
can be used to enhance the functional properties of proteins. In physical modifica-
tion, proteins are subjected to controlled heating and shear conditions that lead to
unfolding or partial denaturation of these macromolecules [140, 141]. Chemical
modification involves acylation, sulfitolysis, phosphorylation and alkylation,
which alters the secondary, tertiary and quaternary structure of proteins alongwith
their hydrophilicity-hydrophobicity balance [142–144]. Enzymatic modification
is an effective approach to enhance the functionality of proteins by means of
hydrolysis and polymerization reactions catalyzed by proteases (pepsin, chymo-
trypsin & trypsin) and transglutaminases. The controlled hydrolysis generates
smaller oil droplets than intact proteins and also increases the emulsifying activity
index[145, 146].
.. Hydrocolloids
For achieving stability in the non-dairy milk alternatives, the addition of
hydrocolloids like guar gum, locust bean gum, Gum Arabic, carrageenan, xanthan
gum, and so on, is often carried out to prevent creaming and phase separation
[147–149]. The charge on polysaccharides impacts their ability to inhibit the
aggregation of oil bodies or fat droplets as well as of proteins by the formation of a
protective coating around them. For instance, carrageenan, an anionic hydrocol-
loid, adsorbs to cationic regions on surfaces of aggregating proteins and hence,
prevents aggregation near their isoelectric point by creating strong electrostatic
or steric repulsive forces [150]. However, studies suggest that while hydrocolloids
are capable of promoting stability at high concentrations, they tend to create
instability in emulsions at low concentrations. Different mechanisms have been
hypothesized to elucidate this phenomenon. When two droplets covered with a
surfactant are in close vicinity, a link between the droplets develops which creates
a connection between droplets [151, 152]. Development of numerous contacts
of this type tend to encourage flocculation and increase the creaming rate. This
is generally identified as “bridging flocculation,” and it is more common when
the hydrocolloid is a weak emulsifier [153]. Therefore, the success of emulsion
stabilization depends on the choice of proper biopolymers that lack the attraction
to the dispersed phase droplets.
Other mechanism, proposed as “depletion flocculation,” was initially suggested
by Asakura and Oosawa [154, 155] and was supported by many scientists later on
[156–158]. According to mechanism, upon addition of any nonadsorbing hydrocol-
loid to a reasonably concentrated emulsion, elimination of the hydrocolloid might
occur in the area between droplets, because of its hydrodynamic size, and thereby
leads to development of local osmotic pressure gradient. The osmotic force results
in the aggregation of oil droplets. The extent of the attractive force is related to the
molecular weight and conformation of the hydrocolloid and varies proportionally
with the concentration of the nonadsorbing hydrocolloid. Such kind of instability
may be prevented by mixing the polysaccharide in less quantity so that aggregation
does not occur.

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4.2 Advanced processing techniques in relation to stability of non-dairy milk
alternatives
.. Ultra high pressure homogenization (UHPH)
Ultra high pressure homogenization is an emerging technology which can be
utilized to enhance the stability of plant based milk alternatives by reducing the
colloidal particles. UHPH produces more uniform sized particles and improves the
physicochemical characteristics of food products without affecting their nutritional
properties [159]. Apart from reducing the particle size, this technique can also
be applied to improve the shelf life of plant based milk alternatives by means of
simultaneous destruction of microorganism [160]. UHPH involves the use of high
pressure in the range of 200–600MPa and temperatures between 30 and 85°C
[161]. The use of UHPH also displays an important role in reduction of allergenic
character of plant-based milk alternatives. Briviba etal. [21] investigated the effect
of UHPH treatment (350MPa at 80°C) on physicochemical properties of almond
milk. No significant reduction in vitamin B1 and B2 contents was reported while the
mean particle size increased threefold. A reduction (99.8%) in almond protein anti-
gens response was also observed. The effect of UHPH treatment (300MPa at 80°C)
and UHT treatment (142°C, 6s) on microbiological, physical and sensorial proper-
ties of soymilk was evaluated [162]. The study showed a reduction in hydroperoxide
index and microbial growth throughout the storage period for both treatments.
Slight differences in sensory characteristics were observed between treatments;
however, panelists did not consider these differences to compare treatments.
.. Pulsed electric field (PFF)
Pulsed electric field is another promising technology that involves the use of
short electricity pulses to inactivate microorganisms in food products while causing
minimal changes in color, flavor, taste and nutritional components [163]. In this
technology, food is placed between two electrodes and electric fields (5–50 KV/Cm)
are generated with the help of short high voltage pulses (microseconds) between the
electrodes. The voltage range can be used for development of non-dairy milk alterna-
tives according to the requirements of size reduction. The experiment carried out by
Xiang [164] investigated the effect of pulsed electric field treatments with different
electric field intensities and number of pulses on structural modification and rheo-
logical properties of soymilk. Pulse electric field treatments at electric field intensi-
ties (18, 20 and 22kV/cm) and number of pulses (25, 59, 75 and 100) increased the
apparent viscosity of soymilk (6.62 to 7.46) as compared to control (not treated).
The changes were attributed to the PEF induced coagulation of the soy protein and
reduction in size of fat globules and their distribution in soy milk. Similarly, Cortes
etal. [165] explored the impact of treatment time (100–475μs) and electric field
intensity (20–35kV/cm) on the quality attributes of horchata (a Spanish vegetable
beverage) during 5days at refrigerated storage (2–4°C). The study revealed that PEF
treatment significantly decreased the peroxidase activity and a negative correlation
was found among peroxidase activity and pH. The increase in pH was proportional
to increasing treatment time in the same electric field intensity.
.. Ultrasound processing
Ultrasound processing is an effective non-thermal technology applied for
processing and preservation of foods. Ultrasound processing is based on the

Milk Substitutes - Selected Aspects
16
phenomenon of acoustic cavitation i.e. rapid expansion and contraction of bubbles
of gas/vapors. This generates intense local heating and high pressures that causes
disintegration of microbial cells and reduces the size of colloidal particles as well.
In the study conducted by Iswarin and Permadi [166], the effect of ultrasound on
droplet diameter of coconut milk was evaluated. The beverage was subjected to dif-
ferent combinations of power levels (2.5 to 7.0W) and exposure times (5 to 25min-
utes) and a reduction in particle size of coconut-based milk was observed as the US
power and time increased. Similarly, Maghsoudlou etal. [19] studied the effect of
ultrasonication treatment on physical stability of almond milk when applied at a
power level of 300W for the time periods of 0, 2.5 and 5min. It was revealed that
exposure time for 5minutes was sufficient to manufacture a desirable product.
The study demonstrated a decrease in sedimentation tendency of milk as well as
decreased viscosity of almond milk. The improved stability has been attributed to
cavitation induced fragmentation of colloidal polysaccharide molecules into smaller
size particles. Size reduction of plant cellular material keeps them in suspension and
hence, aids in improved stability.
5. Future prospect and conclusion
Being a fast-growing segment of food market, the plant-based milk substitutes
need to be extensively explored by using advanced processing and innovative
technologies to produce a nutritionally complete beverage with high overall
acceptability. Plant-based milk substitutes lack cholesterol, milk allergens, lactose,
antibiotics, and saturated fatty acids that make them convenient to be considered
nutritious, economical, health promoting, palatable dairy-free beverage. To meet
consumer’s needs, it is essential to produce high quality beverages having good
physical stability and desirable sensory attributes. Addition of stabilizers and
processing are crucial steps in determining the stability and shelf life of plant-
based milk alternatives. Manufacturers and consumers are more interested in
clean label options for use as additives. Since synthetic stabilizers are generally
added for improving the stability of milk substitutes, the natural substitutes
could present a plausible solution to consumers. Some advanced food processing
techniques including ultra-high pressure homogenization, pulsed electric field
processing, ultrasound processing and high pressure processing can be employed
to overcome instability factors responsible for limiting success of these beverages.
Progressive efforts are required for improving product quality through research and
development activities.

17
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DOI: http://dx.doi.org/10.5772/intechopen.96376
Author details
JyotikaDhankhar* and PreetiKundu
Department of Food Technology, Maharishi Dayanand University,
Rohtak,Haryana, India
*Address all correspondence to: jyotika.ft@mdurohtak.ac.in
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/
by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

18
Milk Substitutes - Selected Aspects
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