Milk protein based encapsulation of probiotics and other food material: comprehensive review

ABSTRACT

Encapsulation plays a vital role in the food industry, known for its multifunction constituents’ preservation, covering undesirable food components (taste, color, flavor), nutritional and functional components incorporation, and under controlled conditions (time and rate) release of encapsulation ingredients. Milk proteins are highly demanding encapsulating material and are investigated at large scale to design encapsulating devices. Polyphenols, flavorings, fatty acids, minerals, and hydrophobic vitamins are within encapsulating bioactives. Milk protein is widely used in the microencapsulation (ME) of probiotics and in comparison to other biomaterials offers more benefits. Milk protein includes whey protein and casein and several techniques are developed to make use of them in the ME of multiple probiotic strains (Bifidobacterium, Lactobacillus). This review will cover all the possible aspects of dairy proteins and their properties that enabled them to be used as encapsulating material. This review will also discuss the range of hydrophobic and hydrophilic components delivered from milk protein and their utilization in the formation of encapsulating agents. Overall, this study aimed to explore the emerging role of encapsulation in the food industry, characterize milk proteins as effective encapsulation agents, investigate their synergism with nondairy encapsulating materials, and evaluate various encapsulation techniques.

KEYWORDS:

• Milk protein
• encapsulation
• Food matrix
• emulsification
• spray drying
• Food applications

Introduction

Encapsulation is an extensively used process in food industries focusing on the manufacture of engineered products, more specifically for functional food processing and product innovation. Owing to the extensive demand for functional foods (probiotics, omega-3, phytochemicals, and vitamins), functional compounds are integrated into the escalating amounts of food products such as yogurt drinks, chocolate, peanut butter, and meat, fruit juices, milk, bread, and tortillas. Microencapsulating technology in terms of industrial application getting more attention because of its potential to preserve unstable bioactive ingredients introduce new operative properties into certain food products and release the material into the targeted controlled area. Consequently, for a long time, numerous techniques have been explored. It is determined by the type of the material (active and shell material) and the expected final product qualities. A wide range of materials is used as encapsulating matrices, polysaccharides, lipids, and proteins are a few of them. Milk proteins are highly effective encapsulating ingredients, considering their physicochemical characteristics which are used in encapsulants in food products.^[1] Milk proteins contain emulsifying, gelling, viscosity-building, film-forming, and soluble properties, and based on the proper and suitable techniques their functional properties can be further modified and broaden their usage in multiple applications as encapsulating matrices.^[2] Apart from that milk protein in combination with other food-grade materials can also be used in the designation of encapsulated food materials (probiotics, hydrophilic and hydrophobic components). Similarly, probiotics can be used solely or in combination with other biomaterials for microencapsulating agents. On the other hand, milk protein is also known to be a good choice for the micro and nano-encapsulation of nutraceuticals and probiotics.^[3–6] The present review is devoted to milk protein as encapsulating material, its properties, processing techniques in food material and probiotics encapsulation, and the combination with other biomaterials. Furthermore, the paper seeks to focus on the specific application of milk protein-based encapsulation and present a promising avenue for addressing challenges related to environmental degradation and the preservation of bioactive compounds in the food industry.

Encapsulation as an emerging technology used in food material

The European Commission reports that the pharmaceutical and cosmetic sectors have invested significantly in encapsulation research and development for the goal of controlled release of bioactive components because of their greater financial resources compared to the food industry.^[7] Comparatively, in 2013, only 1.3% of firms in the food and beverage industry spent any of their revenue on research and development, while the pharmaceutical industry spent 14.4%. Companies in the food business, for instance, are limited by the requirement for mass production of consumables. This raises questions regarding the benefits and drawbacks of the enclosing device. Using dietary proteins in the encapsulation, storage, and distribution of bioactive chemicals is a major driver propelling the development of this market Figure 1.^[8–11]

Figure 1. Encapsulation of different food ingredients using proteins.

Encapsulation is a technique for encapsulating sensitive bioactive chemicals and microorganisms in tiny capsules. Encapsulation has enormous potential in the food industry for the creation of novel and innovative foods with essential uses.^[12] By incorporating the bioactive functional components, microorganisms, and other materials into the core of an encapsulation device, one of the primary goals of encapsulation is to protect against harmful environmental aspects such as light, temperature, oxygen, or moisture, this can be accomplished through the process of encapsulation.^[13] As the food sector continues to adopt additives and bioactive components, the market for encapsulated food products is likely to rise. Encapsulation can preserve sensitive food additives and bioactive compounds (such as probiotics, omega-3 oils, and delicate flavors) against environmental deterioration (Quirós et al., 2014). Moreover, encapsulation can protect food additives and bioactive compounds from unwanted interactions during processing and storage (reducing, for instance, the oxidation-causing interactions with unsaturated oils).^[14] Various studies aim to manage the food components release at the correct place and time, increase the bioavailability of bioactive compounds, and conceal the disagreeable smells of functional food ingredients (such as peptides and polyphenols).^[15–17] Patent literature devotes a significant amount of space to describing how milk proteins can be used as encapsulating components for various culinary additives. This highlights the interest in encapsulated food ingredients, which may be formulated using a wide variety of food ingredients like milk proteins.

Characterization of milk protein

Water, proteins, lactose, fat, and inorganic particles make up the bulk of milk’s physiologically complex fluid composition. Milk consists mostly of water. Primarily milk proteins are divided into two forms, (caseins and whey), and can be found in varying concentrations throughout a wide range of milk species. Casein makes up 60% of the protein in human milk, whereas whey makes up 80% of the protein in cow, sheep, goat, and buffalo milk, and 50% of the protein in equine milk.^[18] Casein micelles are a potential component of milk. Caseins Sl-casein (Sl-CN), S2-casein (S2-CN), -casein, and k-casein combine to form micelles. The milk that we drink contains micelles. Whey is a source of four different types of protein: β-lactalbumin (β-lac), β-lactoglobulin (β-lg), blood serum albumin (BSA), and immunoglobulins (Ig). Other than these, whey fraction counts for over 60 native enzymes, as well as proteoses and peptones (PP), serotransferin, lactotransferin, osteopontins, Lactoferrin, and vitamin binding proteins.^[19,20] Research is conducted on the chemical make-up, nitrogen-based compounds, and the protein fractionations of the casein and whey proteins found in the milk of a variety of dairy animals including buffalo, cows, sheep, goats, and camels. In comparison to the contents of other types of milk, buffalo, and sheep milk have a higher proportion of fat, solids that are not fat, and total solids (Table 1). The percentage of whey protein found in milk from cows is the lowest, while the percentage found in sheep’s milk is the highest (0.54%). Milk proteins are adaptable encapsulating components that may be used alone or in conjunction with other food materials to create microencapsulated food ingredients.

Table 1. Different protein percentages in the milk of different Animal Species.

Animal Species	Whey protein%	Casein protein%	Crud protein%	True protein%
Cow	0.54 ± 0.02	2.58 ± 0.02	3.60 ± 0.02	3.12 ± 0.01
Camel	0.61 ± 0.01	2.46 ± 0.02	3.42 ± 0.02	3.11 ± 0.02
Buffalo	0.73 ± 0.01	2.93 ± 0.02	4.20 ± 0.02	3.667 ± 0.02
Goat	0.55 ± 0.02	2.57 ± 0.02	3.45 ± 0.02	3.12 ± 0.02
Sheep	0.78 ± 0.02	4.38 ± 0.02	5.76 ± 0.01	5.1 ± 0.01

Milk protein as encapsulation agent in food

Using the appropriate processing techniques, researchers can change or enhance the functional qualities of milk proteins, because of this, they may be applied in more applications, such as encapsulation matrices. Therefore, it is generally agreed that protein hydrogels are superior materials for ME in food, especially in meals that are either liquid or semi-solid. They can build matrices of varying sizes without sacrificing the food’s texture or flavor.^[21] Owing to the functional groups that they contain, proteins may also protect, interact, and reverse-bind with many other types of active chemicals. Additionally, they may have useful texture-stabilizing qualities. Milk proteins are widely acknowledged as a vital dietary element due to their well-documented physiological and nutritional characteristics.^[22]

Due to milk proteins’ structural and functional diversity, they can be utilized to transport and encapsulate bioactive substances. Milk proteins are effective interfacial agents for emulsions containing hydrophobic bioactive compounds and can transport hydrophobic chemicals or ions. With target molecules and gel bioactive chemicals, they can form covalent or electrostatic connections (Table 2).

Table 2. Encapsulation of different food ingredients using milk protein.

Food ingredient	Milk protein-containing encapsulation	Source
Bacterial cells	Gelled whey protein matrix containing active entrapped inside of it	^[Citation23]
Oil-containing polysaccharide FA	Whey protein and reducing sugar were combined into an oil emulsion, and the dispersion was heated at 80C for 30 minutes before being spray-dried	^[Citation24]
Lipid-based material	Active emulsified with protein	^[Citation25]
Lipid with colorants/flavor	Active in an oil emulsion that contains a calcium ion and a component that forms a gel, such as milk protein	^[Citation26]
Lipophilic nutrient	Cationic protein complex coacervate that is active-encapsulated	^[Citation27]
Food grade substance	Active entrapped in denatured proteins	^[Citation28]
Antioxidant	One biopolymer around the liposome and another biopolymer with an opposing charge.	^[Citation29]

In addition to their emulsion-stabilizing properties, milk proteins also play a crucial role in hydrogel-based encapsulation systems by inducing the formation of a gel phase. This gel phase serves as an effective medium to incorporate various food components, expanding the scope of applications for such systems. In the case of coacervate-based encapsulation systems, the behavior of milk proteins takes an interesting turn. These proteins interact with biopolymers possessing opposite charges, resulting in the formation of a distinct phase characterized by encapsulated components ensconced within it. This interplay of opposing charges contributes to the selective encapsulation of various substances within the coacervate phase.^[16] Because of their significant interactions with a wide variety of bioactive compounds, they are also capable of performing the role of carriers. Milk proteins offer an extra advantage over other encapsulating materials in that they can easily be utilized in a variety of drying procedures. This is one of the many aspects in which milk proteins show more benefits than other encapsulating materials.

Many different milk protein products are sold in stores. There are many different types of milk proteins available, each with its own unique composition, functional properties, and protein content. These include acid and rennet caseins, caseins and whole milk protein, β -casein, caseinate salts, whey protein concentrates and isolates, and β-lactoglobulin(β-LG) and β-lactalbumin(β-LA) rich fractions. That opens up a wide variety of possibilities for encasing probiotics in milk proteins. Any form of hydrophilic, hydrophobic, or live probiotic cell can be encapsulated in milk proteins.

For the ME produced by living bacteria to be efficient, both the type and the conditions of gel formation are essential. Milk proteins, when subjected to conditions that are quite moderate, are capable of forming gels in many ways. This provides a wide range of opportunities for the efficient utilization of milk proteins inside the ME of living bacteria.^[30,31] When used in capsulation, concentrated milk protein solutions form high-density gels that offer great protection for the probiotics that are encapsulated. These solutions have moderate viscosities, which enables facile bacterial cell dispersion. Milk proteins include a high concentration of bioactive peptides, which have a wide variety of impacts on the body’s physiological systems. These helpful bioactive peptides are produced as a result of the action of digestive enzymes, which is subsequently followed by the positive effects themselves. The bioactive peptides that have been liberated may have an effect that is synergistic with the action of the probiotic.

Microencapsulation based on whey protein

Proteins derived from whey are globular in shape and have an extensive variety of roles in the body. The mammary gland is the only organ in the body that is capable of producing the two primary whey proteins that can be extracted from cow’s milk: lactoglobulin and lactoalbumin. Whey protein has a long history of being renowned as a high-quality wall material, particularly for use in the encapsulation of PUFA-rich oils and subtle flavors. When compared with other proteins, such as soy, this protein is significantly more important in terms of encapsulation efficiency (up to 89.6%). Spray-dried microcapsules were stable for 60 days while maintaining high water activity (aw = 0.74–0.90). Milk proteins are good for carrying oil-soluble actives because they stabilize oil-in-water emulsions. Whey protein isolate (WPI) or whey protein concentrate (WPC) can microencapsulate (AMF).^[32] Therefore, it is now possible to acquire milkfat in powdered form. When compared to a nondairy-based polymeric matrix, WPC alone performed exceptionally well in encapsulating (CLA) conjugated linoleic acid in gum arabic and a WPC maltodextrin blend.^[33] (Jimenez et al., 2006). In addition, wall compositions that are based on WPI and WPC offer remarkable defense against lipid oxidation.^[34]

β-Lactoglobulin (β-LG) is the most prevalent whey protein in cow’s milk. It’s a hydrophobic nano-carrier. This molecule’s hydrophobic calyx binds cholesterol, retinoic acid, vitamin D, aroma chemicals (aldehydes and ketones), and (FA) fatty acids (CLA, palmitate, and oleate).^[34,35] Spray-drying protein-stabilized oil-in-water emulsions were used to test the encapsulating characteristics of WPC, this was done so that we could figure out how to best provide the necessary levels of safety.^[36] In vitro, the WPI matrix acted as a barrier between the retinol and the gastric fluid, which enabled the retinol to be discharged into the environment of the intestinal fluid. The water-soluble fragrance molecule 3-methylbutyraldehyde is stable when encapsulated with WPC, which demonstrates good encapsulation capabilities.^[37] Whey proteins’ exceptional gelling and emulsifying characteristics were put to use in a different application, protecting fat-soluble, bioactive retinol by emulsification and cold gelation before drying (Table 3).^[38] Encapsulation was achieved in that study by spray drying a double emulsion (W/O/W) of water in oil in water, which included a solution of 3-methylbutyraldehyde. Also, whey powder is an effective transporter for acidic flavors.^[39]

Table 3. Whey protein application as an encapsulating agent on different food components.

Whey protein type	ingredient to be Encapsulating(core)	Encapsulation benefits	Source
Whey protein concentrate (WPC)	Carotene	Protection against oxidation	^[Citation40]
	Oregano, marjoram and citronella	During spray drying Improved retention of the flavours	^[Citation32]
	Caffeine	Caffeine release in a controlled manner	^[Citation41]
	CLA	Protection against oxidation	^[Citation42]
Whey protein isolate (WPI)	Orange oil	Protection against oxidation	^[Citation43]
	AMF	Oxidation Protection	^[Citation44]
	Flaxseed oil	Protection against oxidation	^[Citation45]

Microencapsulation, which involves the twofold emulsification of whey proteins and/or gelation of those proteins, has proven to be an effective method for shielding proteins from potentially hazardous aspects of their natural environment.^[46] Water soluble cores such as caffeine also use microencapsulation.^[47] In every case, the gels or microcapsules that were produced were subjected to either air drying or freezing drying before the functionality of the encapsulant system was evaluated.

Microencapsulation based on casein protein

Caseins are a form of protein that precipitates at a pH of 4.6. Caseins are typically considered to be intrinsically unstructured proteins due to their precipitation point.^[48] Micelles of casein are highly hydrated colloidal particles that are generated in milk when caseins react with water.^[49] Casein micelles are made up of around 94% caseins and 6% minerals, the bulk of which are calcium phosphate. This percentage is based on the casein micelles’ dry weight. When it comes to spray-drying dairy-based oil-in-water emulsions, sodium caseinate is an excellent encapsulate to use. Whey protein, on the other hand, is not as effective. There is a wide variety of possible oil-to-protein ratios that may be accomplished with sodium caseinate, anything from 0.25 to 5.^[50] Higher fat powders had poorer microencapsulation effectiveness due to less protein covering of fat globules before drying. This is due to caseins’ high surface activity and heat resistance. Sodium caseinate is used to manufacture spray-dried powders and oil in oil-in-water emulsions. Powders have several uses. ^{[51] [52]} Sodium caseinate in addition is an excellent choice for use as a wall material to preserve orange oil for microencapsulation. In a novel microwave-assisted technique, casein was utilized to encapsulate citric acid. Curcumin, a naturally occurring bioactive with anticancer characteristics, can be delivered utilizing the casein micelle as a delivery vehicle for hydrophobic chemicals.^[36,53] Several biopolymers were examined to see whether or not they were able to enclose citric acid and make high-quality microcapsules. Casein has exceptional film-forming properties, which led to the production of fine powders that featured crystals that were uniformly covered and smooth all over. These crystals were effectively encapsulated. However, when compared to inulin-coated microcapsules, in chewing gum casein-entrapped citric acid was proven to be of lower quality (Table 4).^[54]

Table 4. Casein protein application as an encapsulating agent on different food components.

Casein protein type	ingredient to be encapsulated	Encapsulation benefits	Source
Sodium caseinate	Soy oil	Excellent encapsulation efficiency	^[Citation51]
Casein	Citric acid	Cost-effective and higher encapsulation efficiency	^[Citation36]
Sodium caseinate	3-methylbutyr-aldehyde	During storage better retention of aldehyde	^[Citation53]

Milk protein synergism with other non-dairy encapsulating material

Proteins exhibit greater sensitivity toward structural changes with their efficacy as wall materials depend highly on various external factors just as temperature, and ionic strength (of solutions, etc.^[55,56] Minimizing adverse environmental effects on the functionality of proteins (to be used as encapsulate) can be better achieved by blending them with many other substances significantly the carbohydrate-based biopolymers i.e., lactose, maltodextrin, and corn syrup solids.^[57] In dairy-based emulsions formulated with carbohydrate-protein blends, the proteins in milk show great film-forming as well as emulsifying properties while the carbohydrates mainly act as matrix-forming substances or filler.

For the preparation of nondairy, as well as dry-dairy-based emulsions, anhydrous milkfat (AMF) and lactose are extensively used by combining it with whey protein (WP) or caseinate. To improve the efficiency of using active magnetic fields (AMF) as encapsulating agents, it is possible to achieve this by adding lactose to a wall system primarily composed of sodium caseinate for spray drying. This observation highlights that when subjected to dehydration, lactose demonstrates a robust glassy state. In a separate investigation, the inclusion of lactose was discovered to play a beneficial role in the encapsulation process within emulsions of soy oil that were stabilized using whey protein and then subjected to spray drying.^[50,58] Young et al.^[32] compared the effectiveness of microencapsulating properties of 37% lactose, 10% lactose, and WPI (0% lactose). Regardless of the high AMF loadings i.e., up to 75% wt, WPC50 also exhibited have greater lactose percentage that resulted in enhanced microencapsulating properties. Another research by Keogh and O’Kennedy,^[57] illustrated that a significant decrease in free fatty contents of microencapsulated AMF was observed by increasing the lactose: WPI ratio but surface fat content did not decrease in the same way even after this modification. The stability of microencapsulated-lipids formulations toward oxidation was increased by utilizing alternative carbohydrates to lactose hydrolyzed starches i.e., corn syrup solids, maltodextrin, and dried glucose syrup^[59,60] or trehalose^[61] and combining these with proteins. It was found that stability during storage can be increased by replacing lactose with some other carbohydrates such as maltodextrin or gum Arabic in protein-based emulsions (spray dried) but after reconstitution, it results in increased particle size content, during spray drying, the mixture of maltodextrins and WPC used to enhance the volatile retention and improve stability toward oxidation of microencapsulation, an opposite effect was observed by utilizing mixtures of SMP and maltodextrin, thus decreasing oxidative stability and volatile retentions.^[62] Products of Maillard reactions produced by heating reducing carbohydrates and casein are significant for oil encapsulation that are sensitive to oxidation such as However, utilization of whey proteins for carrying complex and sensitive bioactive compounds depends on the development of cold set gels, which provides fascinating opportunities for the formation of whey proteins as carriers for heat sensitive and hydrophilic bioactive substances. Water soluble substances can also be encapsulated using the coacervation technique. For example, thiamin entrapment can be carried by WPI pectin coacervates.^[63 Some examples of mlk protein encapsulation in combination with other materials has been illustrated in (Table 5).

Table 5. Examples of milk Protein encapsulation in combination with other materials.

Milk Protein	Non-dairy material	ingredient to be encapsulated	Benefits	Source
Whey protein isolate	Alginate	Riboflavin	Controlled release of riboflavin	^[Citation64,Citation65]
Low lactose milk whey	Maltodextrin	Beta-galactosidase	Better retention of β-galactosidase activity during freeze-drying and storage	^[Citation46]
Whey protein isolate	butter fat, corn oil	Lactoferin	Enhanced antibacterial action retention	^[Citation46]
Whey protein isolate	Pectin	thiamine	High thiamine entrapment	^[Citation66]
Casein	Pectin	Lactobacillus and Bifidobacterium sp.	Enhanced cell viability in in vitro gastrointestinal	^[Citation67]
Lactose	Milk proteins	Bifidobacterium sp.	Increased cell viability while being stored	^[Citation68]
Lactose	Milk proteins	Bifidobacterium sp.	Increased cell viability after exposure to gastrointestinal stimuli	^[Citation69]

During the process of drying, different food materials can be mixed with dairy proteins to shield enzyme activities by entrapment within its matrix (amorphous). The activity of enzymes can be better retained by adding maltodextrin. This can also lead to interrupt lactose crystallization and browning in powders while storage at different relative humidity levels (RH).^[70] The food industry is now focusing on microencapsulation of flavors and aromas that are of main concern these days. For this purpose, blends of carbohydrates and proteins of whey have been considered to be efficiently used as carrier matrices. Such as microencapsulation of ethyl caprylate and ethyl butyrate in wall systems along with WPI or 1:1 ratio of WPI: lactose. Factors depending on the retention of volatile substances: wall solid concentration, initial ester load, wall and ester types. For preventing cracks on the surfaces of microcapsules (that were spray dried) mixture of WPI with carbohydrates (with high DE) proved to be more effective than with carbohydrates having low DE.^[71] Core protection can be highly maintained only in case surface cracks are absent. This can also lead to prevent deterioration or/and losses of encapsulated material during storage.

Milk protein-based encapsulation of probiotics

Probiotics can be defined as “Live microorganisms which when administrated in adequate amounts confer a health benefit on the host live microorganisms.”^[72] Probiotics are potential bacteria well known to play a vital role in the human intestinal tract by eradicating harmful microorganisms from the intestine and strengthening the immunity of the human body.^[73] Bacteria are found to be highly sensitive to environmental factors like moisture, oxygen, acidic environment, and temperature and therefore they need to be protected against unfavorable conditions come upon during different techniques. Over the last decade, probiotic demand has climbed up worldwide. Global expenditure is steadily increasing on account of supplements, ingredients, and food of probiotics as it was 21.6 and 24.32 $ billion US dollars in 2010 and 2011 respectively, and it is predicted to reach 31.1 $ billion US dollars in 2015, thus reaching a growth percentage of 7.8% annually.^[74] The diameter of microbial cells normally ranges from 1–5 µm and this property makes them easily entrapped by ME. ME is destined to provide a barrier for protection between them and various damaging environmental conditions i.e., oxygen, moisture, and heat. Important factors to be kept in mind while encapsulating probiotics: probiotics must be viable until they approach the desired site and efficient removal of entrapped microorganisms. It is observed that several areas in the gastrointestinal tract could be targeted by probiotics and all the cells must be released in those specific areas.^[75]

Milk proteins are most likely to be used singly or by combining them with different biomaterials for capsule materials for probiotics. Dianawati et al. ^{[76] [77]} depicted that milk proteins are proved to be a better option for protecting Bifidobacterium longum 1941 in comparison to soy protein isolates against unfavorable conditions i.e., bile or acidic environment and also after freeze-drying. Probiotic encapsulation can be better performed by utilizing biomaterials such as whey proteins (WP) because these proteins are capable of enhancing probiotic resistance against bile salts and acids (especially Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus).^[71] Lactobacillus casei BNCC 134415 were encapsulated by Li et al.. (2019) under lyophilization conditions by using encapsulating materials such as WPI, gellan gum, and cellulose acetate phthalate. It was observed that immobilized probiotic viability is better enhanced during pasteurization, cold storage, and under gastrointestinal fluid. Studies found that the incorporation of 1 or 2% alginate, chitosan, xanthan gum, WPC (50% protein), and L-carrageenan into the medium affects the viability and growth of strains of Bifidobacterium and Lactobacillus.^[78] Bifidobacterium Bb12 viability was studied in whey microcapsules at 4°C during storage. The spray drying method giving rise to microcapsules showed constant and high viability for more than 3 months. Probiotic count proved to remain greater than 7 log CFU/g in dairy desserts for about 6 weeks if microcapsules were added.^[79] During storage and processing activities probiotics are protected by protein gels and probiotics application in the food industry is also extended.^[80] In addition, proteins are proven to be more significant nutritionally than the polysaccharide-based system. Particularly milk proteins are vital nutritionally and possess many bioactive attributes. Enzymatic-induced gelation are means by which casein-based microcapsules are prepared for probiotic encapsulation with the help of rennet^[30,81] or by using transglutaminase.^[31,82] Encapsulation of L. rhamnosus GG, Lactobacillus F19, and Bifidobacterium Bb12 was also performed this way.^[17] In classical storage circumstances, casein encapsulation was proved to increase the survival of cells up to 90 days. The efficient release of probiotics (encapsulated) in the intestine is also a significant property to be considered. Under acidic environments and storage conditions barrier properties were provided by carrier materials but the point to ponder is that probiotic cells should be able to digest microcapsules when released in the gut. This release provides better interaction of probiotics within intestinal microbiota. Microcapsules are mainly produced by singly WP^[83] or a combination of whey proteins and caseins^[81] that during incubation in simulated gastric juice can prevent breakdown (having pepsin) but in intestinal fluid, it can be hydrolyzed fully within 3 hours.^[83] Additionally, in comparison to alginate capsules it was found that protein capsules are preferable (since their protein structure is hydrolyzed during ingestion).^[84] It was concluded that probiotic microorganisms can be better protected by microcapsules (made by milk proteins) against detrimental environmental factors and for effective approach in the human intestine.^[21]

Milk protein synergism with other non-dairy materials for the encapsulation of probiotics

The literature discusses the safe and effective use of probiotics within casein carbohydrate conjugates, demonstrating their suitability for spray drying in oil-in-water emulsions.^[85] In vitro studies showed that the probiotic cells, specifically Bifidobacterium infantis, could be released in simulated intestinal fluid while being shielded from digestion in simulated stomach fluid. Skim milk was identified as an ideal material for encapsulating bifidobacteria, allowing their survival in the gastrointestinal environment and during storage of spray-dried microcapsules.^[68,69]

Another approach explored the utilization of coacervates formed between anionic polysaccharides and casein at pH values below the casein’s isoelectric point. This technique was applied to microencapsulate probiotics (Bifidobacterium lactis and Lactobacillus acidophilus) using spouted bed drying.^[67] Although a slight decrease in probiotic viability occurred (0.3 log drop) after drying, the microencapsulated bacteria were not effectively protected in low-pH environments.

For extended storage (42 days at 4°C), encapsulation in skim milk demonstrated the most favorable matrix for maintaining the viability of Bifidobacterium long B6 and Bifidobacterium infantis CCRC 14,633 populations when compared to other investigated wall materials (such as soluble starch, gum Arabic, and gelatin).^[68] To produce freeze-dried probiotics, various disaccharides, reconstituted sweet whey powder (SMP), and disaccharide combinations were tested for their ability to enhance lactobacilli survival after storage at room temperature under different relative humidity levels.^[86] Disaccharides, particularly trehalose and a combination of lactose and maltose, were identified as effective agents for enhancing cell viability during freeze-drying and subsequent storage at low humidity levels (0–11.4% RH).

The potential of whey protein and other biomaterials was also demonstrated. Spray-dried Lactobacillus rhamnosus GG was used in the matrices that contained whey proteins and various starch ratios. These mixtures were added to apple juice and stored at 4 and 25°C for 5 weeks. All formulations including whey proteins alone or in combination with starch provided L. rhamnosus GG in apple juice or citrate buffer with improved protection as compared to the formulation containing only starch. The capacity of whey proteins to provide a buffered environment within the particle can be used to explain this result. In these circumstances, the bacteria are shielded from the rigors of apple juice’s low pH.^[87]

Techniques for the encapsulation of probiotics based on milk protein

In recent times, consumer acceptance of functional foods with positive metabolic activity has increased dramatically. For this purpose, innovative trends in the research and development sector are observed to produce a variety of food applications with positive health perspectives. A prime example of innovation is encapsulation, in which healthy probiotic cells are sealed inside a protective shell to shield them from harmful elements including moisture, light, air, and temperature. Additionally, this technique is employed to maintain the probiotic cell’s viability during storage, commercialization, and incorporation into food items. This method’s primary goals are to maintain cells throughout the gastrointestinal system even at the low pH of the stomach (2–3) and to release the bacterial cells under controlled conditions at the appropriate location. The coating material utilized to encapsulate the substance that is intended to maintain and release at the correct target location while traveling through the unfavorable gastrointestinal GI tract is known as shell/matrix and is also referred to as coating shell.^[88,89]

In the realm of commerce, diverse encapsulation methods find application in the creation of capsules housing probiotic cells. This study comprehensively addresses the prominent encapsulation technologies, encompassing an array of methodologies like extrusion, fluidized bed coating, spray drying, coacervation, emulsification, and gelation induced by transglutaminase. Encapsulation for the controlled release of bioactive chemicals was initially investigated and used by the pharmaceutical and cosmetic sectors, but it also became quite popular in the food business due to its effectiveness and capacity to accomplish certain goals. The development of encapsulation technologies for creating tiny, water-insoluble microcapsules to protect probiotics from menacing environments is a key objective because milk proteins are well-known biopolymers with the correct physicochemical characteristics for use as food encapsulates, they are a viable encapsulating material.^[90,91] Figure 2 shows the probiotics encapsulation techniques

Figure 2. Probiotic encapsulation techniques.

Extrusion

Microencapsulation of viable probiotic cells to produce microcapsules using hydrocolloids as encapsulating stuff under high pressure through the application of a needle is commercially produced by using the extrusion process. This process is done by using a nozzle to extrude a concentrated mixture of viable probiotic cells and suitable protein-based solution in the form of droplets which then fall into a hardening solution and the hard-gelled droplets are generally known as microbeads. Probiotic cells are first placed inside biopolymers (milk protein, gelatin, and zein) during the extrusion process, which can be used to declare the coating shell as recognized as safe (GRAS). Next, the material is allowed to pass through a nozzle, and movement through vibration is applied to achieve homogenization in size, after which these droplets are instantly solidified in capsules using chemical or physical means.

The choice of biopolymer for encapsulation plays a key role in achieving the desirable characteristics (size, shape, and density) in the final product.^[89] Extrusion is a suitable process because of the formation of large-sized beads that resist the harsh acidic conditions during stomach digestion.^[92] However, protein-based biopolymers are preferred to prevent rapid degradation in an acidic environment. Moreover, the beads were rapidly degraded in the presence of digestive enzymes. Probiotic release from whey protein-based capsules is hypothesized to be controlled by the gel structure rather than proteolysis.^[93,94]

Extrusion-based production of the microcapsule is a suitable process to preserve the sustainability and functioning of probiotic cells. Shi et al.^[95] used alginate milk to entrap Lactobacillus bulgaricus through the extruder application. The nozzle and alginate concentrations were utilized to determine the size and form of the beads. With nozzle sizes of 0.45 and 0.20 mm, beads measuring 830 ± 10 and 381 ± 8 μm respectively were produced. The diameter of the beads became larger when the alginate concentration was raised, and the structure became more spherical.^[96] Moreover, 100% results were achieved in gastric digestion during the trial study.

Emulsification

An emulsion technique is achieved by incorporating an immiscible liquid into another liquid and the addition of probiotics during the emulsion formation leading to the creation of microcapsules.^[97,98] This method is also used to entrap probiotics, in which a mixture of whey protein and live probiotic cells are emulsified in vegetable oil with the help of an appropriate surfactant or emulsifier to form a single emulsion. The double emulsion can be created by homogenizing the single emulsion in an aqueous phase that contains an appropriate surfactant. Emulsification produces capsules in a range of different sizes. The emulsion technique is currently widely used in the culinary, pharmaceutical, and biotechnology industries due to its broad variety of commercial applications.^[99,100]

Lactobacillus acidophilus is encapsulated by gelation employing mixes of alginate and whey protein because the particle size of the probiotic cells is crucial for imparting the desired features of materials. For culinary applications, particles of a size between 33 and 180 μm are appropriate. Additionally, given the challenging stomach and intestinal circumstances, encapsulation increased the survival and viability of the encapsulated cell. The utilization of vegetable oils for the emulsion formation, process is more economical and reliable for the industrial sector.^[101,102] Probiotics have been effectively protected using microencapsulation of whey proteins that include emulsification from harmful circumstances like heat, pH, and enzymes to release the cores on the target side in a regulated manner.^[103,104]

Spray drying

One of the most well-known microencapsulation processes is spray drying, due to the rapid evaporation of water as well as to maintain the low temperature in the encapsulation material which makes it unique among other techniques. Before the drying process, pre-drying treatment is given to the material including the growth of bacterial strain, encapsulation in biopolymer as a coating, and spray drying is a multistep process that takes place before initiating the process of spray drying, the mixture is gently stirred to attain the homogenized encapsulated components.^[105,106]

Spray drying is significant due to its better survival rate of probiotic cells and improved resistance during gastric transit. However, specific influential factors can jeopardize the proper functioning of the probiotic cells due to adverse conditions in the gastrointestinal environment.^[107] Moreover, the selection of appropriate bacterial strains is crucial before drying because some probiotic strains cannot attain their viability due to heat, pH, and osmotic stress. Spray drying is responsible for some viability damages in encapsulated cells, related to physical damage to the microbeads, and bacterial cell release, due to heat creation during the drying process. Keeping in mind the problems associated with probiotic viability, scientists demonstrate the conditions in which bacterial strains can survive. The process includes atomizing a probiotic emulsion in a carrier material to drying gas that leads to rapid evaporation of moisture.^[106]

Aeration of probiotics in emulsion has been demonstrated to provide protective conditions for probiotics that are encapsulated by casein-carbohydrate conjugates. In different in vitro studies, probiotic cells were subjected to and assessed their stability against the highly acidic environments of the stomach and another intestinal environment. Microencapsulation of Bifidobacterium animalis by Loyeau et al.. (2018) using the spray drying method was trailed in gastrointestinal digestion and observed that bacterial cells were negatively affected in the digestion process with a storage capacity of 12 months. In another investigation by Loyeau et al.. (2018) whey proteins by heating, it has been shown that Bifidobacterium encapsulated using spray drying had improved the subsistence of encapsulated probiotic cells during the simulated stomach digestion. The method is entirely repeatable, and perhaps most importantly, it can be used in industries.

Coacervation

A homogeneous solution of polymer is coacervated into two phases: coacervate (a polymer-rich phase) and equilibrium phase (a polymer-poor phase. Sacervus, which means waste in Latin, is the root of the Latin term acervus. Separating colloidal particles from the fluid and depositing them on a core is the main goal of this approach. Coacervate nuclei develop a uniform distribution all around core particles during this process, adhering to the surface of the core material. In conclusion, the cross-linking of the phases by a chemical, enzymatic, or thermal process solidifies the coating material.^[108,109]

Coacervation is a physicochemical method for creating polymeric microcapsules, which are often used in medicines and cosmetics. Coacervation is a common technique used nowadays to encapsulate probiotic organisms and maintain their viability (Table 6). In complex coacervation, a kind of complex formation, electrostatic interfaces play a major role in the parting of a biopolymer-rich phase. In certain climatic conditions, interacting biopolymers form insoluble complexes that lead to the development of two distinct liquid phases: a lower phase that is high in biopolymers, particularly complex coacervates but is low in biopolymers.^[110] Systems with intricate coacervation, such as proteins and ionic polysaccharides of opposed charge, are fascinating. Popular anionic polysaccharide pectin has been used in complicated coacervation with β-lactoglobulin. The coacervates’ storage circumstances may cause gradual structural alterations.

Table 6. Milk protein-based encapsulation of probiotics using different encapsulation techniques.

Milk protein	Probiotics	Encapsulation technique	Source
Caseinate	Bifidobacterium sp.	Spray drying	^[Citation85]
Whey protein coat	Lb. acidophilus	Extrusion	^[Citation111]
Rennet whey	B. animalis BB-12	Spray drying	^[Citation79]
Whey powder	Lb. reuteri	Fluidized bed	^[Citation112]
Whey/canola oil	Lb. rhamnosus	Emulsification	^[Citation113]
Whey protein isolate	Lb. plantarum	Coacervation	^[Citation114]
Whey protein concentrate	B. animalis BB-12	Electrospraying	^[Citation115]

Milk protein encapsulated probiotics applications in food ingredients

Functional ingredients are the major concern of food processors due to their low stability during processing and consumers’ increasing acceptance is increasing day by day due to their health-promoting properties.^[116,117] Many foods are enriched with functional ingredients (probiotics and antioxidants) but they are lost during harsh processing conditions. Functional ingredients are used by food manufacturers to preserve food’s color, taste, texture, and fragrance as well as to increase shelf life. Researchers tested the microencapsulation of several probiotic strains efficiently with increased stability to overcome the stability and vitality of probiotics.

Probiotics are beneficial bacteria that are unable to survive in a crucial gastrointestinal environment. However, different studies and their industrial implementation reveal the importance of microencapsulation. It is used on a larger scale and covers almost every field in the food industry including dairy, beverage, meat, and bakery. Probiotics are used for their health-promoting effects however due to the harsh intestinal environment their viability is a major concern for researchers. In different studies, their applications are being studied with technological advancements.

The viability of Lactobacillus rhamnosus, when encapsulated in whey protein (WP) beads, was compared against free cells that were freeze-dried using a milk-based solution and a denatured whey protein isolate (WPI)-based solution with added lactose and sucrose. This comparison was conducted during the production and storage of various products, including biscuits, frozen cranberry juice, and vegetable juice. When biscuits were processed and stored at a temperature of 23°C for two weeks, the decline in cell counts was least pronounced in the samples encapsulated using whey protein. Similarly, Lactobacillus rhamnosus, when microencapsulated in a combination of WPI and resistant starch (RS), demonstrated better preservation in apple juice or citrate buffer compared to when it was encapsulated in RS alone. This was observed during a five-week storage period at both low and ambient temperatures.^[118]

The beneficial impact of whey protein isolate (WPI) on the survival of entrapped probiotics is attributed to the protective environment it creates around the cells, guarding them against external challenges. In separate experiments, auxiliary cultures and encapsulated whey protein yogurts were prepared using Bifidobacterium breve and Bifidobacterium longum. The selection of the encapsulation technique and strain played a significant role in the encapsulation yield and viability, as indicated by Abd El-Salam et al.^[121] B. breve exhibited a higher encapsulation yield with a survival rate of 25.77 ± 0.1%, compared to 1.47 ± 0.2% for B. longum when subjected to spray-drying, a method recommended due to its heat stability. Encapsulated B. breve within yogurt demonstrated significantly higher viable counts than free cells after 28 days of storage at 4°C, while no protective effect was observed for B. longum. In terms of various matrices, free cells embedded in a milk-based matrix showed the highest viability during storage in vegetable juice, as well as during the freezing and storage of cranberry juice. Furthermore, Bifidobacterium Bb-12 microencapsulated using spray drying with whey maintained a probiotic count exceeding 7 log CFU.g − 1 when incorporated into a dairy dessert over six weeks.^[79]

Conclusion and future trends

As encapsulation emerges not only as a tool for health promotion but also as a means of extending shelf life and enhancing sensory attributes, its adoption in the food industry is poised for continued growth. The integration of additives and bioactive components, coupled with the rising market for encapsulated food products, underscores the relevance and timeliness of this research. The characterization of milk proteins, particularly whey and casein, as effective encapsulation agents has been highlighted. The synergistic interactions between milk proteins and nondairy encapsulating materials have been discussed, shedding light on the potential for enhancing stability and functionality. The focus on probiotic encapsulation using milk proteins has revealed promising outcomes, with potential benefits in health promotion. The various encapsulation techniques, including extrusion, emulsification, spray drying, and coacervation, have been evaluated for their applicability in preserving probiotics. Probiotics can be trapped and microencapsulated in milk proteins for a variety of reasons, and in most cases, this increases the likelihood that they will survive the digestive process. Optimizing the utilization of milk proteins in microencapsulation necessitates basic research in various domains. Because live cells react differently to various biopolymers, further research is still required to determine how milk protein encapsulation affects food components. To generate the capsulated product on an industrial scale, which turns into the final product inexpensively, standardization of the proposed microencapsulation procedures and subsequent pilot plant and industrial-scale manufacturing are still needed. Large-scale manufacturing and administration of microencapsulated probiotics would allow for a more accurate in vivo assessment of survival of probiotics and health benefits in humans .

Acknowledgement

We are thankful to all the collaborators and members of the Food Safety and Biotechnology lab.

Disclosure statement

No potential conflict of interest was reported by the author(s).