*Article* **Fatty-Acid Profiles, Triacylglycerol Compositions, and Crystalline Structures of Bambangan-Seed Fat Extracted Using Different Solvents**

**Norazlina Mohammad Ridhwan 1, Hasmadi Mamat 1,\* and Md Jahurul Haque Akanda <sup>2</sup>**


**Abstract:** Currently, research on the bambangan-fruit seed has become interesting because of its potential application as a cocoa butter alternative. This work aimed to determine the changes in the quality of the extracted bambangan-seed fat (BSF) obtained using hexane, petroleum ether, and ethanol. The extraction solvents affected the total fat content (TFC), physicochemical properties, fatty-acid profile, triacylglycerol composition, and crystalline structure of the extracted BSF. The results showed that BSF has a high content of 1,3-distreoyl-2-oleoyl-glycerol (SOS). The solvent-type significantly (*p* < 0.05) impacts the stearic and oleic acids of the extracts, resulting in apparent changes in the high-melting symmetrical triacylglycerols, such as SOS. Petroleum-ether-extracted BSF has a high stearic acid of 33.40%, followed by that of hexane- and ethanol-extracted BSF at 29.29% and 27.84%, respectively. Moreover, the spherulitic microstructure with needle-like crystals of the extracts also ranges from 30 to 70 μm in diameter. Hexane-extracted BSF illustrated a less-dense, spherulitic, crystalline microstructure with a less-granular centre than those extracted using the other solvents. The results suggested that the quality of the extracted BSF obtained from the nonpolar solvents of hexane and petroleum ether are better than that extracted using ethanol.

**Keywords:** bambangan; extraction solvents; fatty acid; triacylglycerol; crystalline microstructure

## **1. Introduction**

*Mangifera pajang* is an indigenous fruit distributed around the Borneo Islands, such as Kalimantan (Indonesia), Sabah and Sarawak (Malaysia), and Brunei [1]. This fruit is locally known as bambangan and has become a prominent, underutilised fruit with significant economic value. Bambangan trees can grow up to 30 m tall, with a cylindrical bole with smooth, broadly fissured, grey bark [2]. It initially grows widely in the forest and is currently cultivated by the local Kadazan–Dusun people, specifically in Sabah [3]. The cultivation of bambangan fruit in Sabah was reported as having a constant growth of 121.6 to 133.03 metric tons from 2016 to 2020, as the trees are currently being planted in orchards or in the backyards of homes, corresponding to the increasing demand for this fruit [4,5]. Bambangan fruit is larger in size, and it has a thick peel (of a brown colour, with rough skin), fibrous flesh. Each fruit can weigh up to 1.5 kg [6].

The local community prefers mature bambangan fruit for consumption and utilises this fruit in functional food-forms, including juice and processed fruit, and as a health drink, and it can be added to food as a flavouring ingredient. However, the seed is not consumed, but rather disposed of as a waste by-product. This waste by-product has been reported to have significant health benefits, based on the considerable number of antioxidant compounds found in the seed and in the peel [2,4,7]. The seed is made up of 9.8–11% fat, 3.08–4.1% protein, and 38.68–72.9% carbohydrate, indicating that the seed has nutritional potential as a source of protein and carbohydrates [7–11]. Bambangan-seed fat (BSF) is

**Citation:** Ridhwan, N.M.; Mamat, H.; Akanda, M.J.H. Fatty-Acid Profiles, Triacylglycerol Compositions, and Crystalline Structures of Bambangan-Seed Fat Extracted Using Different Solvents. *Appl. Sci.* **2022**, *12*, 8180. https://doi.org/10.3390/ app12168180

Academic Editor: Alessandra Biancolillo

Received: 15 July 2022 Accepted: 12 August 2022 Published: 16 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

mainly composed of palmitic (7.29–15.8%), stearic (32.37–40.39%), oleic (39.24–48.05%), and linoleic (4.95–8.11%) fatty acids (FAs), which corresponds to the presence of three main triacylglycerols (TG): SOS, SOO, and POS (8.7–40.70%, 11.20–26.87%, and 11.60–11.93%, respectively) [1,9,12,13]. BSF has also become an interest for researchers due to its similarities with cocoa-butter-like fats: illipe butter, mango-seed fat, kokum butter, sal fat, and shea butter [4,8,10,12,14]. Moreover, BSF is SOS-rich, which makes it applicable as an SOS-rich fat resource to increase the hardness of soft fats, which is desirable in a country with a high climate.

The extraction of BSF can be performed in various ways, including using Soxhlet extraction. Soxhlet extraction is economical, simple, and allows several extractions to be carried out simultaneously with high oil-recovery as compared to the other method [15]. The operational cost is also lower because the solvent can be recovered after the extraction, creating reusable solvents to be used for another extraction process [16]. Soxhlet extraction is an economical method that lowers operational costs by using reusable solvents with higher extraction efficiencies than the other method. Using different solvents in Soxhlet extraction gives variation to the oil-quality parameter and thus could extend the applicability of the oil based on its properties, and it offers the best option for extraction. Moreover, fresh solvents are repeatedly brought into contact with the sample, thus supplanting the equilibrium transfer [17]. The extraction's efficacy depends on the temperature, oil nature, particle size, sample pre-treatment conditions, time, and solvent type [18]. The choice of solvents for the extraction is essential for determining the quality of the extracted fat. Different studies have reported on the ways that extraction solvents influence oil quality, specifically the yield and bioactive compound levels [17,19,20]. The process of the Soxhlet extraction of oil can be performed using ethanol; the polar protic solvents or hexane and petroleum ether; the nonpolar solvents [20].

Hexane is commonly preferred among solvents due to its low-melting properties, high availability, and polarity, which lead to high solubility [21–23]. In comparison, petroleum ether has been used for the extraction of lipophilic compounds, and ethanol has been used because of its low-toxicity properties and high availability, as well as its being bio-based [24,25]. Hexane has been classified as a toxic chemical by the US Environmental Protection Agency because it can react with air pollutants to produce ozone and other environmental pollutants [26]. Hence, it is only permitted in maximum amounts of 5 ppm and 10 ppm in meal and oil, respectively, under the PFA Act of 1954 [27]. Several replacement solvents have been found to extract oil from oilseeds without utilising hexane due to safety, health, and environmental concerns [28,29]. Thus, hexane substitutes, for instance, ethanol, water, petroleum ether, and other potential solvents, have been developed and used for oil extraction [25].

However, the work of comparing the fatty acid (FA) composition, triacylglycerol (TG) content, and crystalline microstructure of BSF, as extracted using different solvents, is still in the early stage. Thus, this study aimed to evaluate the changes in the physicochemical properties (iodine value and Slip melting point), FA and TG compositions, and the crystalline microstructure of the extracted BSF using different solvents, as well as the efficiency of the extraction solvents.

## **2. Materials and Methods**

## *2.1. Materials*

Ripe bambangan fruits were provided by a local farmer in Ranau, Sabah, Malaysia. The following items were acquired from Sigma–Aldrich: acetone, acetonitrile, cyclohexane, ethanol, hexane, n-hexane, methanol, potassium hydroxide, petroleum ether, potassium iodide, sodium thiosulfate, starch indicator, Wijs solution, triacylglycerols, and fatty acid methyl esters standard. The analytical chemicals, reagent-grade chemicals, and extraction solvents used were of the highest possible quality.

#### *2.2. Extraction of Bambangan-Seed Fat (BSF) Using Hexane, Petroleum Ether, and Ethanol*

Each bambangan seed was separated from the flesh and then cut into small pieces (10 mm × 10 mm × 5 mm) for sample preparation. Next, it was stored in a drying cabinet (48 h at 45 ◦C) for drying processes. Each dried seed was ground into a powdered form using a grinding mill and kept at −20 ◦C before the analysis. The extraction was conducted using the AOAC [30] official method of analysis for Soxhlet extraction (Method 945.16), using petroleum ether with slight modifications. A total of 80.0 ± 0.00 g of seed powder was extracted for 8 h at 40 ◦C using 3 different solvents: hexane, petroleum ether, and ethanol. Ethanol was used as a hexane substitute for oilseed-extraction because of health, safety, and environmental concerns [28,31]. The remaining solvent in the extracted BSF was removed using a rotary evaporator (40 ◦C) (HEIDOLPH LABORTA 4001) and then filtered in an oven (at 45 ◦C) to remove any impurities. The total fat content (TFC) for the fat is expressed as the following equation:

$$\text{TFC } \left( \% \right) = \frac{\text{Extracted cycle fat (g)}}{\text{Bambangan seed power (g)}} \times 100 \tag{1}$$

#### *2.3. Physicochemical Properties*

The changes in the physicochemical properties, such as the iodine value (IV) and the Slip melting point (SMP), of the extracted BSF were determined according to the AOCS [32] official methods, Cc 3b-92 and Cd 1b-87, respectively. For IV analysis, 0.5 g of melted BSF (at 60 ◦C) were homogenised with 20 mL of cyclohexane and 25 mL of Wijs solution (iodine solution) and left in the dark for 1 h. Next, 20 mL of 15% KI and 100 mL of distilled water were added to the mixture. A quantity of 0.1 N sodium thiosulfate solution was used to titrate the mixture. After the yellow solution became colourless, 2 mL of the starch indicator was added, and the mixture was titrated until the blue solution became colourless. The following calculation was used to calculate the IVs of the fat samples:

$$\text{IV} \left(\text{g}\,\text{iodine}/\text{g}\right) = \frac{\left(\text{Vol}\,\text{of}\,\text{blank}\,\text{tit}\,\text{rat}\,\text{t} - \text{vol}\,\text{of}\,\text{sample}\,\text{tit}\,\text{rat}\,\right) \times \text{Normality}\,\text{of}\,\text{tit}\,\text{rat}\,\times\text{12.69}}{\text{mass (g)}}\tag{2}$$

The SMP of the hexane-, petroleum-ether-, and ethanol-extracted BSF was determined using an open-ended capillary glass tube. Before analysis, the glass tube was dipped into the fat samples to a depth of 10 mm, and the fat was chilled and solidified in an ice bath. Using a rubber band, the BSF samples were attached to the bottom of the thermometer and then immersed in the glass test tube before being placed in a water bath (10 ◦C) for analysis. The hot-plate temperature (SP131320-33-V, Thermo Scientific, Shanghai, China) was gradually increased by 1 ◦C to increase the water-bath temperature until the fat column ascended. When the fat column reached a height of 30 mm, the SMP of the fat samples was determined.

## *2.4. Profile of FA*

The FA content of the extracted BSF was determined using a gas chromatography– flame ionisation detector (6890 N, Agilent, Santa Clara, CA, USA) as described by Norazlina et al. [9]. The FA methyl esters (FAMEs) for the extracted BSF were prepared before being injected into the BPX70 column (30 m × 0.25 μm × I.D. 0.25). A quantity of 0.5 g of BSF was dissolved using 2.5 mL of n-hexane and 0.5 mL of potassium hydroxide in methanol (2 N), vortexed (1 min at 1200 rpm), and left to stand at room temperature. After 10 min, the translucent upper-layer was injected into the GC for analysis. The following condition was used to identify the FAMEs: an initial temperature of 90 ◦C (hold for 5 min), then raise by 8 ◦C at a time to 185 ◦C (hold for 1 min), then raise by 2 ◦C to reach a final temperature of 250 ◦C (hold for 5 min). Using split-mode, maintain a temperature of 250 ◦C for the injector and detector (1:20). The FA profile was determined using the FAMEs standard. The results were presented in % concentrations and compared with the FAME standard.

#### *2.5. TG Content*

The TG composition of the extracted BSF was measured according to AOCS [32] official method Ce 5c-93, using high-performance liquid chromatography (HPLC; 1200, Agilent, Mississauga, ON, Canada) equipped with a refractive index detector (RID) with slight adjustments. For sample preparation, 0.1 g of the melted fat samples (at 60 ◦C) was diluted to 10 mL of mobile-phase solution (acetone: acetonitrile, premixed) to make a 10% solution. The mixture was then filtered through a 0.45 μm PTFE syringe filter (47 mm millipore diameter) and placed into the HPLC vial for analysis. A C18-HPLC column (Kromasil C18, Merck, Germany) was used for the study. An injection volume of 5 μL, a column temperature of 30 ◦C, a detector temperature of 40 ◦C, a pressure of 8–9 mPa, and mobile-phase acetone: acetonitrile (70:30, *v*/*v*) were utilised in the studies. The results were presented in % concentrations and compared with the TG standard

## *2.6. Crystalline Structure*

The changes in the crystalline structure for the 3 extracted BSFs were observed using polarised light microscopy (DM2500P, Leica, Wetzlar, Germany), a method developed by Narine and Marangoni [33]. The crystalline structure of the fat crystals helps define the texture of a product for use in confectionery because it is directly related to the polymorphic behaviour of a fat [34]. A quantity of 15 μL of melted BSF was placed on the microscopic slide (heated at 80 ◦C), covered with a coverslip, and then chilled at 4 ◦C for 1 h. The samples were then incubated at 25 ◦C for 2 days for proper crystallisation before being observed under a polarised light microscope at 40× magnification.

## *2.7. Statistical Analysis*

The total fat content (TFC) analysis and all other studies were conducted in triplicate, and the results are expressed as means and standard deviations (±). The Tukey test and one-way analysis of variance (ANOVA) were used to find any significant differences in the treatment means. A *p* < 0.05 significance value was used to define the significance level.

#### **3. Results**

#### *3.1. Extraction of BSF and Its IV and SMP Properties*

Figure 1 shows the differences in the physical appearance of the extracted BSFs obtained using hexane, petroleum ether, and ethanol. BSF extracted using a nonpolar solvent is lighter in colour than is polar-solvent-extracted BSF. The hexane and petroleumether extracts showed similar appearances, with a common yellow oil-colour, and they solidified faster than did the ethanol extracts. A similar observation was reported in the extraction of kariya seed oil: nonpolar solvents produced yellow oil extracts, and the polar solvent produced a cloudy, dark-golden oil extract [35]. The variation in the extracted BSF appearances was presumably associated with the acid value and free-fattyacid content, in which free fatty acid is more soluble in the polar solvent [36]. Therefore, the ethanol-extracted BSF had a darker-golden appearance than the hexane and petroleumether extracts.

The TFC, IV, and SMP properties of the extracted BSFs are shown in Table 1. The solvents selected, such as hexane and petroleum ether for the current study, are typically used to extract oil from plant kernels [17], while ethanol is considered a green solvent in chemical extraction because of its low toxicity. Among the extraction solvents, hexane produced a high TFC, followed by the petroleum-ether and ethanol extracts. It can be seen that the hexane had a higher level of efficiency for extracting the BSF. The solutes and solvent interactions, boiling temperature, and solvent polarity might be the prominent factors that influenced the BSF's yield, extraction efficacy, and composition [17]. The hexane's low-polarity properties caused rapid molecule-transfer between the solvents, thus leading to a high TFC in the hexane-extracted BSF as compared to the petroleum-ether- and ethanol-extracted BSFs [37]. The presence of the antioxidant compounds and other extract compounds, soluble primarily in polar solvents, presumably led to the low yield of TFC in the ethanol-extracted BSF. The extraction yield for the 3 extracts was comparable to the reported fat content of mango seed fat, with values of 5.73–7.74% in a previous study [38]. On the other hand, the extracted BSFs' physicochemical properties, such as the IV and the SMP, also showed variation in their values.

**Figure 1.** Physical appearance of the extracted BSFs using hexane, petroleum ether, and ethanol.

As seen in Table 1, petroleum-ether-extracted BSF has the lowest IV, followed by the hexane- and ethanol-extracted BSFs. The IV is used to determine the unsaturation levels and the stability of oil for industrial applications [39]. A low IV in the petroleum-extracted BSF indicates that it is prone to better resist oxidation, and it has a higher quality and longer shelf-life than the other two extracts. This behaviour is supported by the results of our previous study, in which the petroleum-ether extracts with low IVs showed a low acidity, with a value of 3.74 mg KOH/g, and had better thermal profiles than the ethanol extracts, with a 7.41 mg KOH/g acid value [40]. The changes in the IV also are associated with the FA composition. A low unsaturation value indicates a low presence of unsaturated FA (UFA); thus, petroleum-ether extracts have more saturated FA (SFA) than hexane and ethanol extracts. The extraction solvents also influence the SMP for the extracted BSFs. The petroleum-ether-extracted BSF showed high SMP values followed by the hexane and ethanol extracts. The low IVs in the petroleum-ether extracts indicate more SFAs and a higher melting-point-TG content than the other solvents; thus, the SMP is higher. The trends in the efficacy of the extraction solvents and the changes in the physicochemical properties for the extracted BSF align with the results reported by Kittiphom and Sutasinee [17] and Jedidi et al. [20].



Values are the mean ± standard deviation of three replicates; means with a different letter (a, b, or c, with a showing the highest value) within a column are significantly different (*p* < 0.05) as measured by the Tukey test.

## *3.2. Characterisation of FA Profiles in BSF Extracts*

Vegetable fats and oils are beneficial for industrial and food purposes, and their quality is closely related to their FA composition [20]. As shown in Table 2, significant (*p* < 0.05) changes in the FA profiles of the 3 extracts were influenced by the solvents used. Obvious changes can be seen in the extracts' stearic and oleic acid compositions. About 52.32–59.70% of the BSFs' FA composition was dominated by the UFA, primarily oleic (43.90–48.31%), with a significant presence of linoleic (8.04–9.72%) acid. This result explains the high unsaturation value of all extracts, especially the ethanol-extracted BSF. The extracts obtained in this study showed softer properties with high UFA compositions as compared to the BSF reported in the previous study [1], in which 56.19% of the FA composition was saturated. The variation obtained in this study was presumably correlated with the geographical latitude, thus showing variation in their compositions. The composition and quality of the fat may vary depending on the fruit's growth condition. According to Varnham [42], the type of a plant, the environment, and the degree to which the seeds ripen determine the FA and the unsaponifiable components of oilseeds. The bambangan fruit used in the study was ripening but over-softening due to transportation and storage, which made the fruit spoil, resulting in variations in the quality parameter.

**Table 2.** Fatty-acid composition of BSF extracted from hexane, petroleum ether, and ethanol.


Values are the mean ± standard deviation of 3 replicates; means with a different letter (a, b, or c, with a showing the highest value) within a column are significantly different (*p* < 0.05) as measured by the Tukey test.

Additionally, the temperature also significantly impacts the content of FA, particularly the UFA [43]. On the other hand, the results obtained in this study are in agreement with the FA profiles of BSF extracted by Jahurul et al. [12] and Norazlina et al. [9] and the mango-seed fat extracted by Jahurul et al. [44] and Munchiri, Mahungu, and Gituanja [45]. The SFA and UFA for the reported BSF and mango-seed fat ranged from 44.7 to 44.8% and from 29.1 to 58.6%, from 49.2 to 50.2%, and from 41.1 to 70.2%, respectively. Petroleumether-extracted BSF has higher quantities of palmitic and stearic acids, followed by the hexane and ethanol extracts. This shows that petroleum-ether-extracted BSF is harder than the other two extracts. In contrast, ethanol extracts have more oleic acid than do hexane and petroleum-ether extracts. The changes in the FA composition for the 3 extracts can be supported by Kittiphom & Sutasinee [17], who reported similar changes in the extraction of mango-seed oil using hexane (palmitic: 8.97%, stearic: 37.37%, oleic: 43.77%, and linoleic: 6.78%), petroleum ether (palmitic: 8.73%, stearic: 37.70%, oleic: 44.75%, and linoleic: 5.67%) and ethanol (palmitic: 8.50%, stearic: 38.50%, oleic: 43.45%, and linoleic: 6.48%).

Moreover, BSF produced by the Soxhlet extraction in this study exhibited an FA-type similar to commercial cocoa butter (CB) (palmitic: 24.5–33.7%, stearic: 33.3–40.2%, oleic: 26.3–36.5%, and linoleic: 1.7–3.56% acids) reported by Gunstone [46], Sonwai et al. [47], Kadivar et al. [48], and Norazura, Sivaruby, and Noor Lida [49], indicating that the extracts are applicable as potential CB alternatives.

## *3.3. TG Profiles*

The TG fat composition is essential for determining the potential application of the extracts, as well as providing information on the polymorphic behaviour. Table 3 summarises the TG content for all extracts, where it can be seen that the TG content was significantly (*p* < 0.05) affected by the extraction solvents. All extracts were dominated by SOS (30.22–44.29%), SOO (20.19–24.18%), and POS (9.57–12.48%) with a significant presence of OOO (5.18–7.09%), POP (2.44–3.83 %), SSS (1.25–3.40%), OLO (2.90–4.56%), and POL (3.16–3.65%). Based on these values, 39.79–56.77% of the composition is high-melting TG (POS and SOS), thus explaining the solidification process of BSF at an ambient temperature. Petroleum-ether-extracted BSF (56.77%) has a high content of high-melting TG, followed by hexane (48.54%) and ethanol (39.79%). This behaviour results in the high SMP value of petroleum-ether extracts, and it increases the hardness of the fat. The variation in the high-melting composition of the extracts was associated with the solubility of the low-melting TG; low-melting TG is more soluble in a polar solvent [50]. Therefore, the ethanol extracts have more low-melting TG than the other extracts.

In addition, the high-melting TG in the hexane extracts is closer to 50% of the TG composition, thus showing a comparable SMP with the petroleum-ether-extracted BSF. The unsaturation value of the hexane extracts is comparable to the petroleum-ether extracts due to the comparable FA and TG compositions. Therefore, this indicates that the quality of the hexane extracts is comparable to that of the petroleum-ether extracts. The relation between the high-melting TG and the SMP obtained in this study also agrees with the melting point of the POS (23.50–43.0 ◦C) and SOS (19.50–35.50 ◦C), as reported by a previous study [51–53]. The TG content in the study is also in line with the TG profiles of the reported BSFs and mango-seed fats [8,12,13,34,54–58]. The BSF extracted using different solvents also showed a noticeable amount of OLL, PLL, OLO, POL, PLP, and SSS, as in a previous study [12].


**Table 3.** Triacyglycerol content of BSF extracted from hexane, petroleum ether, and ethanol.

Values are the mean ± standard deviation of three replicates; means with a different letter (a, b, or c, with a showing the highest value) within a column are significantly different (*p* < 0.05) as measured by the Tukey test.

## *3.4. Crystalline Microstructure*

Figure 2 shows the crystalline microphotograph of BSFs extracted using hexane, petroleum ether, and ethanol. All fat structures showed spherulite crystals, consisting of needle-like crystals branching outwards with a diameter of 40–70 μm. This structure is commonly associated with the *β* polymorphic form of a CB [59]. Such a crystalline structure is desirable for making chocolate and confectionery products. The crystalline structure of the extracted BSFs was significantly changed after exposure to the extraction solvents. Petroleum-ether- and ethanol-extracted BSFs showed a compact cluster of fat crystal compared to the hexane-extracted BSF. The spherulite structure of the petroleumether-extracted BSF was disrupted after exposure to the solvent, making the structure oval and compact. The ethanol-extracted BSF showed a smaller crystal structure than the hexaneextracted BSF. These findings are similar to the microphotograph reported by Norazlina et al. [9] The changes in the crystalline microstructure (polymorphism, distribution size, size, surface of a structure, and shape) occurred because of the variety in the textural properties of fat and the TG composition [60]. The crystalline microstructure was also potentially influenced by the different FA and TG contents [9], resulting in the difference in the crystallisation state.

(**C**)

(**A**) (**B**)

**Figure 2.** Crystalline microstructure of (**A**) hexane-, (**B**) petroleum-ether-, and (**C**) ethanol-extracted BSF.

Moreover, the saturation-degree of the fat also affected the crystalline microstructure [52]. The unsaturated ethanol-extracted BSF has crystals with a small, densely packed centre, a loose and scattered structure similar to the crystalline microstructure of the mangoseed fat (Thai cultivar) reported by Sonwai and Ponprachanuvut [38]. On the other hand, hexane-extracted BSF exhibited a loosely scattered, spherulite structure similar to that of CB, as reported by Asep et al. [61], thus suggesting that hexane-extracted BSF is similar to CB-like fats and may be applicable as CB alternative.

## **4. Discussion**

This study analysed the FA profiles, TG compositions, and the crystalline microstructures of BSFs obtained using different extraction solvents. To achieve this goal, three different organic solvents were used to obtain the best extraction solvents. Interestingly, the findings showed the solvents affect the composition of the BSF, in which the BSFs extracted using nonpolar solvents (hexane and petroleum ether) are of better quality than those extracted using the polar solvent. Besides that, a previous study also reported that the seed oils extracted using the nonpolar solvents have a high-quality parameter [17,19,20]. The results showed that the characteristics of the extracted BSFs in this study are also in agreement with the reported properties of the BSF reported by Azrina et al. [1], Jahurul et al. [12], and Norazlina et al. [9,13], but the TFC is very low. The unsaturation value (59.84 g iodine/g), UFA (more than 50%) content, and the low-melting TG (33.45%) were high in the ethanolextracted BSF.

The oil's lower solubility could explain the differences in the extracted BSF's quality parameters, such as the TFC, in the polar solvents compared to the nonpolar solvents. The extracted BSF in the polar solvent was mainly disturbed by another component, such as the antioxidant-extract compounds and free fatty acids, which were co-extracted during the extraction process; thus, a low TFC was obtained in the ethanol oil-extracts. The final results, such as the oil yield, FA profile, and the physicochemical qualities, were significantly influenced by the solvents used for extraction, which have been previously reviewed [61,62]. In fact, hexane and petroleum ether, commonly used as the extraction solvents for lipidextraction in seed oils [22,25], are supposed to have similar properties. However, these solvents showed significant differences in the properties of the extracted BSF.

Although petroleum-ether-extracted BSF shows high-quality parameters; it has a low unsaturation value, a high SFA, and a high-melting TG composition, the extracts' crystalline microstructure is dense and oval and has a low yield. Concerning the TFC, FA, and TG compositions and the crystalline microstructure, hexane seems to be the most efficient among the extraction solvents studied, with a TFC of 7.7%. Still, it has a low POP (3.83%) and POS (11.78%) content and a high SOO (23.83%) level, which could result in the softening effect. The toxicity of the hexane solvents could also become a concern, such that the solvents' traces should be analysed and not exceed the permitted maximum amount in edible oil, as mentioned above. Nevertheless, petroleum ether is an alternative solvent choice known to have less toxicity than hexane, and it could also be used as an extraction solvent as it has comparable properties with hexane-extracted BSF but with a significantly lower yield.

## **5. Conclusions**

This report is the first study to explore how different organic solvents affect the composition of BSF. This work successfully performed BSF extraction using polar (ethanol) and nonpolar (hexane and petroleum-ether) solvents. The changes in the physicochemical properties, FA and TG compositions, and the crystalline structures of the BSFs extracted from different solvents are presented in this report. The results suggest that the hexaneextracted BSF had better overall fat quality, including a high TFC and IV and SMP values comparable to the petroleum-ether extracts. Although petroleum-ether-extracted BSF has a stearic-acid content similar to that of CB, the FA profiles are close to those of the hexaneextracted BSF. Both extracts showed FA-types similar to those of CB, and only the hexane

extracts exhibited a similarly less-dense crystalline structure. Therefore, hexane-extracted BSF is applicable as a CB alternative due to its similarities with CB. Therefore, hexane is suitable for the extraction of BSF. This research is beneficial for providing the solvent's choice information for BSF extraction and speciality-fat production.

**Author Contributions:** Data curation, N.M.R.; methodology, H.M. and M.J.H.A.; formal analysis, N.M.R.; software, N.M.R.; writing—original draft preparation, N.M.R. and H.M.; writing—review and editing, N.M.R., H.M. and M.J.H.A.; supervision, H.M. and M.J.H.A.; project administration, H.M. and M.J.H.A.; funding acquisition, H.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors thank Universiti Malaysia Sabah (UMS) Centre for Postgraduate Study and Centre for Research and Innovation for financing this study through the Teaching Assistant Scheme and Grant Scheme: SDN0061-2019.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**Wee Yin Koh 1,\*, Xiao Xian Lim 2, Thuan-Chew Tan 2,3, Rovina Kobun <sup>1</sup> and Babak Rasti <sup>4</sup>**


**Abstract:** The growing health awareness among consumers has increased the demand for non-dairybased products containing probiotics. However, the incorporation of probiotics in non-dairy matrices is challenging, and probiotics tend to have a low survival rate in these matrices and subsequently perform poorly in the gastrointestinal system. Encapsulation of probiotics with a physical barrier could preserve the survivability of probiotics and subsequently improve delivery efficiency to the host. This article aimed to review the effectiveness of encapsulation techniques (coacervation, extrusion, emulsion, spray-drying, freeze-drying, fluidized bed coating, spray chilling, layer-by-layer, and coencapsulation) and biomaterials (carbohydrate-, fat-, and protein-based) on the viability of probiotics under the harsh conditions of food processing, storage, and along the gastrointestinal passage. Recent studies on probiotic encapsulations using non-dairy food matrices, such as fruits, fruit and vegetable juices, fermented rice beverages, tea, jelly-like desserts, bakery products, sauces, and gum products, were also included in this review. Overall, co-encapsulation of probiotics with prebiotics was found to be effective in preserving the viability of probiotics in non-dairy food matrices. Encapsulation techniques could add value and widen the application of probiotics in the non-dairy food market and future perspectives in this area.

**Keywords:** encapsulation; non-dairy; probiotics; stability; storage

## **1. Introduction**

The growing awareness among consumers regarding healthy lifestyles has increased the demand for food that could provide additional specific health benefits beyond nutrition. Functional food is one of the leading trends in today's food industry. The term "functional food" refers to foods containing (either present naturally or added by manufacturers) ingredients or bioactive compounds that provide extra health benefits over its adequate nutritional effects, which can beneficially affect one or more physiological mechanisms in the body, resulting in an enhancement in health and reduction in risk for disease, in the amount consumed in a diet [1]. For example, probiotics are one of the dominant groups of functional foods [2].

Probiotics, from the Greek word, "for life", are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit to the host" by a joint United Nations Food and Agricultural Organization/World Health Organization working group in 2001 and The International Scientific Association for Probiotics and Prebiotics (IS-APP). Probiotics have also been considered functional foods due to their health-promoting abilities [3]. Among probiotic strains in use today, strains from genera of *Lactobacillus* and

**Citation:** Koh, W.Y.; Lim, X.X.; Tan, T.-C.; Kobun, R.; Rasti, B. Encapsulated Probiotics: Potential Techniques and Coating Materials for Non-Dairy Food Applications. *Appl. Sci.* **2022**, *12*, 10005. https://doi.org/ 10.3390/app121910005

Academic Editor: Maria Kanellaki

Received: 8 August 2022 Accepted: 29 September 2022 Published: 5 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*Bifidobacterium* are the most frequently used. In addition, other non-pathogenic microorganisms that occur within the host gut or tissues have also been developed as probiotics. These include strains from genera *Propionibacterium*, *Pediococcus*, *Bacteroides*, *Bacillus*, *Streptococcus*, *Escherichia*, *Enterococcus*, and *Saccharomyces*. Lately, *Faecalibacterium prausnitzii*, *Akkermansia muciniphila*, and *Eubacterium hallii* have also been identified as potential next-generation probiotics with promising health-promoting functionalities [1,4].

By regulating the natural balance of gut bacteria in the human gastrointestinal tract, probiotics have been shown to promote a wide range of health benefits such as improving intestinal health, improving lactose digestion, enhancing the host's immune response, reducing serum cholesterol, diarrhea diseases, and inflammatory bowel disease, counteracting allergies, and lowering the risk of certain cancers [5]. For a potential probiotic strain to exert therapeutic effects on the host, the viability of probiotics in food should be at least 6 to 7 log CFU/mL (or CFU/g) when reaching the small intestine and colon. In this regard, the viability of at least 8 to 9 log CFU/mL (or CFU/g) of probiotics in food before ingestion is necessary [3,6].

Probiotics must be stable throughout the digestive tract and able to adhere to human epithelial cells when they reach the intestine. However, the survival of probiotics is greatly affected by the harsh conditions of the gastrointestinal tract, including the acidic pH of the gastric environment and bile acids (a loss of around 2 log CFU/mL or CFU/g during digestion) [7]. Several intrinsic (e.g., pH, water activity, molecular oxygen, the composition of the food, food additives added, and oxidation-reduction potential) and extrinsic factors (e.g., temperature, relative humidity, and gas composition) have also been observed to negatively affect the viability and stability of probiotics during food preparation and food processing, as well as over a prolonged storage period [5,7,8].

Traditionally, dairy products have been recognized as the best carriers of probiotics. Current probiotics have been formulated into numerous dairy products, such as fermented milk, yogurt, cheese, and ice cream. However, consumers' preferences today lie more with non-dairy-based probiotic products because of the ongoing trend of vegetarianism and awareness of drawbacks associated with the intake of dairy products, such as lactose intolerance, high cholesterol content, and milk protein allergy [2,9]. In recent years, nondairy matrices, such as fruits [10–12], fruit and vegetable juices [7,11–26], fermented rice beverages [27], tea [28,29], jelly-like desserts [30], bakery products [31–33], cereal bars [34], sauces [35], gum products [36], and powdered functional drink [37] have been explored as vehicles to deliver probiotics. Although non-dairy food matrices are more versatile (absent of lactose, dairy allergens, and cholesterol) than dairy food matrices, the delivery of probiotics using non-dairy food matrices is more challenging. As an example of a dairy food matrix, milk, which is rich in proteins and fats, could effectively act as a protective matrix to protect the probiotics throughout the digestive tract [38]. In contrast, non-dairy food matrices, such as fruit and vegetable juices, have considerable amounts of organic acids, dissolved oxygen, and inherently low pH values that could negatively affect the viability of inoculated probiotics [9]. Dairy food matrices are usually stored at refrigerated temperature (4 ◦C), and therefore, the viability of probiotics can be well-maintained throughout the product's shelf life. In contrast to dairy food matrices, non-dairy food matrices are often stored at ambient temperature, which could adversely affect the viability of probiotics [2]. The sensory qualities of non-dairy food matrices could also be enhanced or deteriorated by the metabolic compounds produced through the interaction between the probiotics and food matrices [2,9].

To address these challenges, encapsulation techniques have been implemented to preserve the viability of probiotics. Encapsulation can be defined as "a process in which small solid particles, liquid droplets, or gases are entrapped by a coating layer, or incorporated into a homogeneous or heterogeneous matrix, yielding small capsules with useful properties in immobilization, protection, controlled release, structuration, and functionalization" [39]. In other words, encapsulation is a technique of retaining a substance (core material, such as probiotics) within another (wall material). When applied successfully, the encapsulation technique may improve the resistance of probiotics to the harsh gastric environment and hence, facilitate the controlled release and successful delivery of probiotics to the site of action. By restricting the probiotics from being directly in contact with food components, encapsulation could maintain the viability of probiotics during the food manufacturing process and long-term storage. Through encapsulation techniques, probiotic cultures can be transformed into concentrated dry powder form, which is more stable and easier to incorporate into many food matrices [1,40–42]. This article aims to review and analyze the effectiveness of encapsulation techniques and supplementation of coating materials on the viability of probiotics in non-dairy food and beverage products during storage, as well as while transiting through our gastrointestinal tract.

## **2. Encapsulation**

To date, encapsulation is one of the most promising techniques in protecting active compounds against adverse environments. Encapsulation technology has been widely used in the pharmaceutical, medicine, nutritional, food science, biological, agriculture, toiletries, and cosmetics industries for over 50 years. The goal of encapsulation is to protect the encapsulated active compound (core material) against unfavorable or adverse environments (such as light, moisture, temperature, and oxygen). In food industries, a broad range of products (including probiotics, antioxidants, antimicrobials, flavors, enzymes, and nucleic acids) are encapsulated to (a) prevent the core material from degradation, (b) slow down the evaporation rate of volatile core material, (c) separate the components that would otherwise react with each other, (d) modify the nature of the core material for easier handling, (e) increase the stability, (f) to mask undesired tastes, colors, and odors, (g) enable sustained and controlled release (release slowly over time at a constant rate), (h) control oxidative reactions, (i) use with bacteriophages to control foodborne pathogens, and (j) extend the shelf life. Indeed, encapsulation is one of the new and effective methods to protect probiotics from the harsh conditions they encounter throughout food processing, shelf storage, and gastrointestinal transit [1,40–42].

#### **3. Probiotic Encapsulation Techniques**

Numerous encapsulation technologies have been developed and adopted to protect probiotics. All the techniques aim to protect the viability and stability of probiotics. However, their concepts, operation methods, and properties of produced capsules are different. Each technique also has its own strengths and drawbacks. Figure 1 illustrates different types of probiotics encapsulation techniques and the morphologies of corresponding microcapsules obtained. Various aspects must be taken into consideration before the selection of encapsulation techniques. Selecting a suitable encapsulation technique depends on several parameters, such as the nature of the probiotics, the operational conditions of the encapsulation technique, the properties of the biomaterials used, the particle size needed to deliver the adequate probiotics load without affecting the sensory properties, the release mechanism and release rate, the composition of the target food application, the storage conditions of the food products before consumption, and lastly, the cost limitation of production [43,44].

## *3.1. Extrusion*

Extrusion (also known as external ionic gelation, which produces capsules with sizes of 100 μm to 5 mm) is the oldest and the most common physical technique for encapsulating the probiotic cell. In the extrusion technique, probiotics are first suspended in a biopolymer solution. The suspension is then fed into an extruder (pilot scale) or a syringe needle (laboratory scale) and drips off into a hardening solution (most commonly, calcium chloride) with gentle stirring [40,45].

**Figure 1.** *Cont*.

**Figure 1.** Different types of probiotics encapsulation techniques and the morphologies of corresponding microcapsules obtained: (**a**) ionic gelation (emulsion, extrusion); (**b**) coacervation; (**c**) fluidized bed coating; (**d**) freeze-drying; (**e**) spray-drying; (**f**) spray chilling; (**g**) layer-by-layer method; (**h**) co-encapsulation.

The extrusion technique is relatively simple, direct, straightforward, and gentle (does not involve extreme temperature, pH condition, and organic solvents), thus resulting in relatively high viability (low cell harm) of probiotic microorganisms and requiring a lower operational cost. This technique can be conducted under both aerobic and anaerobic conditions. It is biocompatible and flexible as it does not require any harmful solvents. Using alginate/shellac blend and sunflower oil as wall and core materials, respectively [40,41,43,45], Silva et al. [46] demonstrated that the co-extrusion encapsulation technique increased the probiotic survival of *L. acidophilus* LA3 by about 80% in simulated gastrointestinal conditions and 83% of the probiotics loaded into dried particles were viable after 60 days storage at room temperature (25 ◦C). Kim et al. [47] also demonstrated that encapsulation of probiotic *L. acidophilus* by ionic gelation between phytic acid and chitosan followed by the addition of calcium carbonate and starch with electrostatic extrusion provided buffering effects and protection against acid injury during simulated gastric conditions and refrigerated storage. The extrusion technique has also been utilized in non-dairy probiotic foods such as *E. faecium* in cherry juice [13], *L. lactis* ABRIINW-N19 in orange juice [17], *L. casei* DSM 20011 in pineapple, raspberry, and orange juices [18], *L. acidophilus* TISTR 2365 in sweet fermented rice sap beverage [27], *L. acidophilus* NCFM in mulberry tea [29], *L. casei* Lc-01 and *L. acidophilus* La5 in mayonnaise [35], and *L. reuteri* in chewing gum [36].

However, the size of beads produced through the extrusion technique is relatively big (up to 5 mm), and the process of bead solidification is also relatively slow. Hence, this technique is not suitable to be used in large-scale production [40,43,45]. Over the last decades, an evolving extrusion technique (vibrating nozzle method) has been focused on and studied. This new extrusion technique uses vibrating technology (mechanical principle), in which, when a defined amplitude is enforced, the vibrational frequency will break the extruded fluid into pre-defined-sized droplets. The size of the droplets generated using this technique can be controlled through the diameter of the jet, the velocity of the extrusion process, the viscosity and the surface tension of the fluid, and the frequency of disturbance [41].

## *3.2. Emulsion*

In the emulsion (also known as internal ionic gelation, which produces capsules with sizes of 200 nm to 1 mm) encapsulation processes, the suspension containing probiotics cell and polymer (disperse phase) is first dispersed into vegetable oil (continuous phase) and homogenized to form a water-in-oil (W/O) emulsion in the presence of a surfactant (emulsifier). After emulsification, calcium chloride (cross-linking agent) is added to insolubilize and harden (fast gelling process) the water-soluble biopolymer. The gel beads can then be harvested by filtration or centrifugation [40,43].

Compared to the extrusion technique, the emulsion technique is easier to scale up for mass production. Hence, it is more suitable for application at the industrial level [45]. Additionally, high survival of probiotics was also reported after encapsulation using the emulsion technique [43]. Singh et al. [48] found that probiotic *L. rhamnosus* GG encapsulated in a homogeneous system of carboxymethyl cellulose/gelatin blend survived better under simulated intestinal tract conditions compared to free probiotics. Picone et al. [49] revealed that encapsulated *L. rhamnosus* in gelled water-in-oil emulsions had a survival rate of more than 77% against in vitro digestion. Probiotics (*B. bifidum* [7], *L. acidophilus* PTCC1643, *B. bifidum* PTCC 1644 [15], *L. plantarum*, *L. fermentum*, *L. casei*, *L. sphaericus*, *S. boulardii* [16], and *Lactobacillus salivarius* spp. *salivarius* CECT 4063 [10]) encapsulated using the emulsion technique have also been reported as suitable to be used in non-dairy food matrices such as grape [7,15], tomato, and carrot [16] juices, and apple matrix [10]. Furthermore, the emulsion encapsulation technique is flexible since it can adjust and control the beads' size. According to Oberoi et al. [40], the diameter of the beads produced through the emulsion technique can be reduced to 25 μm, which cannot be achieved using extrusion methods. However, the emulsion technique has a high operational cost due to the high price of vegetable oil (such as soy, sunflower, corn–millet, and light paraffin oil) [40,45]. In addition, the microcapsule produced is not suitable for use in low-fat food products due to the oil residual in the capsule [39].

Considering that conventional emulsions are thermodynamically unstable, advanced emulsion technologies such as nano-emulsion, Pickering emulsion, and Pickering high internal phase emulsion are implemented for efficient probiotics encapsulation. Nanoemulsion is a relatively stable emulsion system with smaller droplet sizes ranging from 50 to 200 nm. In the study by Vaishanavi and Preetha [50], nano-emulsions containing soy protein isolate, Tween 80, and gum Arabic were prepared for encapsulating *L. delbrueckii* subsp. *bulgaricus*. The stability and survivability of the probiotics loaded in nano-emulsions were well-maintained throughout the storage period of 40 days. Contrary to nano-emulsions, Pickering emulsion is an emulsion system that does not require emulsifiers in the stabilization process. Pickering emulsion is stabilized rather by solid particles (more effective with hydrophobic particles). Pickering-type double emulsion (water-in-oil-in-water, stabilized with polyglycerol polyricinoleate) has also been used to encapsulate probiotics (*L. acidophilus*) [51]. The viability (gastric digestion = 93.59%, intestinal digestion = 84.24%) and colon-adhesion efficiency (43.27%) of probiotics entrapped in the double emulsion were higher than the free probiotics (viability after 1 h gastric digestion = 0%, colon-adhesion efficiency = 14.20%) during storage (14 days) and after exposure to simulated gastrointestinal conditions. Pickering high internal phase emulsion is a Pickering-type emulsion with a high internal oil phase fraction. By limiting the probiotics from contact with water and oxygen, Pickering high internal phase emulsion is known to possess high encapsulation efficiency and serve as a promising delivery system for probiotics. In a study conducted by Qin et al. [52], Pickering high internal phase emulsion stabilized with the covalent conjugates of whey protein isolate and (-)-epigallocatechin-3-gallate was used to encapsulate and protect probiotics (*L. Plantarum*). The probiotics encapsulated in the emulsion showed higher viability after storage (14 days) and were more resistant to acidic medium, bile salts, and digestive enzyme digestion when compared to the free probiotics.

#### *3.3. Coacervation*

Coacervation (phase separation, which produces capsules with sizes of 1 μm to 1 mm) is a process whereby an initial solution is separated into a polymer-rich phase (coacervate) and a polymer-poor phase (coacervation medium). Coacervation techniques can be further categorized into simple and complex coacervations. Simple coacervation involves only a single polymer. In simple coacervation, phase separation can be induced when a strongly hydrophilic substance, water-miscible non-solvent, or inorganic salt (desolvation of the polymer) is added into a colloid solution. On the other hand, complex coacervation refers to the ionic interactions between two or more polymers (usually a protein and a polysaccharide) of opposite charges. During complex coacervation, when the charge is neutralized, the polymers separate, deposit on the droplet, and form coacervates [41,53]. Therefore, complex coacervation is preferable in probiotics encapsulation and the food industry [43].

Complex coacervation is known to produce capsules with high loading capacity that can incorporate a high number of probiotics. This technique provides high encapsulation efficiency, even at a very high (99%) payload. Complex coacervation is a simple process that does not involve high temperatures and hence, is safe for probiotics. Sharifi et al. [54] showed that probiotic *L. plantarum* and phytosterols, co-entrapped by heteroprotein complex coacervation utilizing whey protein isolate and gum Arabic, resulted in increased probiotic viability in Iranian white cheese. Silva et al. [46] demonstrated improved resistance to simulated gastrointestinal tract conditions of the microcapsules of probiotic *L. acidophilus* encapsulated by complex coacervation followed by transglutaminase crosslinking (up to 9.07 log CFU/g survival) and maintained probiotic viability (up to 9.59 log CFU/g) for 60 days at freezing (−18 ◦C) temperature.

The complex coacervation technique also produces microcapsules with water immiscibility which leads to optimal controlled-release properties. Complex coacervation can produce microcapsules with sizes ranging from 1 to 100 μm. However, complex coacervation is hard to scale up, as the solute used to form coacervates must be in liquid form. The range of polymers employed in this technique is also limited as coacervates are only stable within a range of pH, ionic strength, and temperature. Gelatin is the most common polymer employed in complex coacervation. However, the use of animal-based protein is limited in certain situations [55]. Zhao et al. [56] demonstrated that in comparison with the protein/polysaccharide complex coacervation, the encapsulated probiotic in water/water emulsion via type-A gelatin/sodium caseinate coacervation had a better survival rate after heating, ambient storage, and simulated digestion. The authors indicated that the increased protection of the type-A gelatin/sodium caseinate matrix was associated with lower hygroscopicity, solubilization, and wettability and could also be caused by the significantly higher hydrophobicity. Complex coacervation is also regarded as a costly technique because an additional hardening process is required.

Complex coacervation is suitable for non-dairy probiotic foods. In the study by Silva et al. [22], probiotic orange and apple juices were produced with the aid of complex coacervation associated with enzymatic crosslinking. As indicated by the results, encapsulated *L. acidophilus* LA-02 incorporated in fruit juices can survive throughout a storage period of 63 days (4 ◦C). In addition, complex coacervation was also used by Holkem et al. [14] to encapsulate *B. animalis* subsp. *lactis* in the development of probiotic sugar cane juice. The viability of *B. animalis* during storage and delivery was enhanced through complex coacervation.

## *3.4. Drying Method*

#### 3.4.1. Spray-Drying

In food industries, spray-drying is the most used method to dry the encapsulated mixture into powdered probiotics (capsule sizes: 5–150 μm). The principle in spray-drying is the simultaneous mass and heat transfer processes between hot air and droplets. There are three main processes involved in the spray-drying process (i) atomization of a solution comprising probiotics and core material into fine droplets, (ii) droplets evaporation in a heated gas stream, and finally, (iii) separation and collection of spray-dried powder [43,44].

The advantages of this drying technique include (i) the process is rapid and continuous, (ii) this technique does not require a high operational cost (10 times cheaper compared to freeze-drying), (iii) highly reproducible, easy for scaling up and suitable for industrial

application, and (iv) the spray-dried products are typically dry, low in water activity, highly stable, and have low bulk density. Studies about the encapsulation of probiotics by spray-drying have been extensively reported. For example, Arslan et al. [57] showed that probiotic *Saccharomyces cerevisiae* var. *boulardii* microencapsulated with gum Arabic, pea protein, and gelatin by spray-drying were more resistant to simulated stomach solution. Jantzen et al. [58] also demonstrated that probiotic *L. reuteri* cultivated in whey slurry microencapsulated by direct spray-drying showed a 32% greater survival rate upon exposure to simulated digestive juice than those without encapsulation. Numerous studies have used spray-drying encapsulation in non-dairy probiotic food. Vivek et al. [20] demonstrated that spray-dried Sohiong juice fermented with *L. plantarum* remained viable (6.12 log CFU/g) for 36 days of storage at 25 ◦C. A study by Hernández-Barrueta et al. [28] showed that the viability of spray-dried *L. rhamnosus* GG in a matrix of whey protein isolate and hydrolyzed extruded huauzontle starch was stable in a ready-to-drink green tea beverage during the 5 weeks refrigerated storage.

The drawback of this technique is the harsh processing conditions, which can cause adverse effects on the stability, viability, and survivability of the probiotics [1,40,43]. For instance, the high temperature and osmotic stress applied during spray-drying can kill the probiotics. Furthermore, high air velocities during spray-drying can result in microcapsules formed with poor uniformity in terms of particle size and morphology.

## 3.4.2. Freeze-Drying

Freeze-drying, also known as lyophilization or cryodesiccation, is a process whereby the water vapor in the frozen sample is removed through the sublimation of ice. This technique produces capsules with sizes of 1–1.5 mm. It is commonly used to preserve thermosensitive components such as probiotics. The process of freeze-drying can be divided into three phases, (i) the initial freezing process of the probiotics (together with the carrier material), (ii) the primary drying (sublimation) phase, and (iii) secondary drying to eliminate the remaining traces of water due to absorption [43].

Rishabh et al. [23] used freeze-drying and spray-drying to encapsulate *E. faecalis* incorporated in carrot juice using gum Arabic and maltodextrin as coating materials. Compared to spray-drying, heat injuries to the probiotics are lower in the freeze-drying technique. Raddatz et al. [59] reported that *L. acidophilus* microencapsulated in the form of emulsification/internal gelation followed by freeze-drying using a blend of pectin micro-particles with prebiotic rice bran maintained probiotic viability for 120 days at 25 ◦C. In another study, Massounga Bora et al. [25] used freeze-drying to encapsulate *L. acidophilus* and *L. casei* using whey protein isolate and fructooligosaccharides as wall material in the development of probiotics-enriched freeze-dried banana powder. During the 30 days of refrigerated (at 4 ◦C) storage, the encapsulated probiotics had higher survivability compared to the free cells. The encapsulated probiotics were also more resistant to simulated gastric intestinal fluid.

However, in another investigation by Shoji et al. [60], the authors did not obtain the same positive findings using microencapsulation of *L. acidophilus* Lac-04 through complex coacervation followed by freeze-drying. The authors observed a significant decrease in viability (*p* < 0.05) after 30 days at 37 ◦C. The microencapsulated probiotics failed to withstand the pH condition of the human stomach. Although the freeze-drying technique has been reported to provide shelf stability to probiotics, sometimes the crystal formation during the freezing process can result in cell injury and eventually lead to cell death. Therefore, cryoprotectants that exert protection for the probiotics are necessary [61]. Cryoprotectants protect probiotics from freezing damage by inhibiting rapid cellular dehydration and ice formation during freeze-drying. Furthermore, freeze-drying is an expensive procedure with high operational and maintenance costs and is not easy to scale up [40].

#### 3.4.3. Spray Chilling

Spray chilling (cooling or congealing, which produces capsules with sizes of 20–200 μm) is like spray-drying, but it injects cold air to atomize and solidify the particles instead of hot air. In the spray chilling process, the encapsulated agent is first dispersed in a molten lipid matrix before atomization in a chamber with cold air injection [62]. In this technique, lipids are utilized as the encapsulation material. During the passage through the gastrointestinal tract, when the temperature reaches the melting point of the carrier material (lipid), the lipases in the intestines digest the lipid wall materials and release the probiotics. Therefore, spray chilling was found to be very promising in the controlled release of probiotics. Spray chilling is reported to be the cheapest encapsulation technique as it provides a high process yield regardless of continuous or batch production. Spray chilling has also been recognized as more environmentally friendly as it requires only mild processing conditions, low operation energy, and time. Since spray chilling does not require heat, the viability of probiotics can be retained. Furthermore, this technique can be operated continuously with the elimination of hold times between manufacturing steps, making it suitable to be scaled up for industrial-scale production [43,48].

*S. boulardii*, *L. acidophilus*, and *B. bifidum* have been encapsulated using the spray chilling technique [63]. The results showed that the survivability of spray-chilled (*S. boulardii*, 97.89%; *L. acidophilus*, 83.57%; *B. bifidum*, 88.50%) and spray-dried (*S. boulardii*, 97.51%; *L. acidophilus*, 84.05%; *B. bifidum*, 90.10%) probiotics under simulated gastric conditions were similar. The spray chilling technique has also been used to encapsulate probiotics in probiotics-enriched cream-filled cakes [31] and savory cereal bars [34]. Spray chilling improved the survivability of *S. boulardii*, *L. acidophilus*, and *B. bifidum* incorporated in cream-filled and marmalade-filled cake samples during refrigerated storage [31]. Similarly, the viabilities of spray-chilled *L. acidophilus* and *B. animalis* subsp. *lactis* were higher than freeze-dried and free probiotics in the savory cereal bars after being stored for 90 days at 4 ◦C [34]. However, low encapsulation capacities on the beads produced through this technique have been reported. A lower load (10–20%) was obtained when compared to spray-drying (5–50%) [64]. The beads produced through the spray chilling technique have low melting points (32–42 ◦C). Probiotics encapsulated using the spray chilling technique were also found to protrude from the beads during storage. Hence, proper handling and storage conditions are required to preserve spray-chilled probiotics [43,44].

## 3.4.4. Fluidized Bed Drying

Fluidized bed drying, or fluidized bed coating, is a modified spray-drying method that involves intensive, simultaneous heat and mass transfers between solid particles in a suspension (produces capsules with sizes of 5–5000 μm). In the fluidized bed drying process, dried pre-encapsulated probiotics are first suspended in a hot air flow. Subsequently, the surfaces of the particles are fluidized with the biopolymer solution. The biopolymer coating is then solidified into a homogeneous layer surrounded by the pre-encapsulated probiotics [44,50]. In the fluidized bed drying process, the aqueous medium is dried in a uniform airflow, and the dried particles are suspended in the heated air. Hence, the particles are evenly dried with much less agglomeration and are uniformly coated. Compared with spray and freeze-drying processes, fluidized bed drying requires less energy consumption, and therefore, it is comparatively economical. Compared to other techniques, a lower drying temperature (ranges from ambient temperature to 120 ◦C) can be set and used in fluidized bed drying. Hence, it can preserve heat-sensitive probiotics. For example, Sánchez-Portilla et al. [65] proved that the viability of *Bifidobacterium* sp. was retained for more than 2 years, with a concentration exceeding 5 log CFU/g, as well as resistance to acid and complete enteric-targeted release, through the fluidized bed drying technique. The fluidized bed drying technique is ideal for food industries as it is easy to scale up and can be prepared in large batch volumes and high throughputs. Fluidized bed drying can provide multi-coating layers. Thus, it can contribute to a variety of functional properties. Nevertheless, this technique is time-consuming (~2 h), likely to kill the probiotics, and

it is not easy to master. Therefore, probiotics should be encapsulated before drying in a fluidized bed dryer [43,44,62,66].

Fluidized bed drying techniques have been successfully used by Galvão et al. [11], Mirzamani et al. [32], and Nilubol and Wanchaitanawong [26] to preserve the viability of probiotics in non-dairy food matrices. Galvão et al. [11] dried and coated apple cubes with a mixture containing hydroxyethyl cellulose and polyethylene glycol containing *B*. *coagulans* using the fluidized bed drying technique. The viability of probiotics in the dried apple snacks was well preserved during the storage period. The fluidized bed drying technique has also been used by Mirzamani et al. [32] to develop probiotic bread. The double-layered (first layer: microcrystalline cellulose powder and alginate or xanthan gum; second layer: gellan or chitosan) microcapsules produced through the fluidized bed drying technique had higher heat resistance and could protect the encapsulated probiotics (*L. Sporogenes*) under baking conditions. In the study of Nilubol and Wanchaitanawong [26], carrot tablets containing *L. plantarum* TISTR 2075 were produced using a fluidized bed drying technique employing gelatin. The finding indicated that the *L. plantarum* TISTR 2075 encapsulated in carrot tablets (survivability: 77.68–87.30%) had higher tolerance against heat digestion treatments than free cells (39.52%).

## *3.5. Layer-by-Layer Method (Multilayer Technique)*

For better performance, encapsulated probiotics are coated with more than one layer, using different polymers for each layer. The layer-by-layer method (multilayer technique) was proven to increase the survivability of probiotics against the conditions of processing, storage, and along the gastrointestinal tract [50,67]. For instance, Beldarrain-Iznaga et al. [68] revealed that microencapsulation of *L. casei* using a combination of layer (canola oil)-by-layer (sodium caseinate) double emulsion and ionic gelation technique could enhance the thermal stability and cell viability of *L. casei* during storage and digestion. The functional characteristics of *L. casei* C24 were also retained through microencapsulation using the layer (alginate)-by-layer (chitosan) double emulsion technique [68]. In another study, layer (carboxymethyl cellulose)-by-layer (zein protein) encapsulating *L. plantarum* 299v was applied to apple slices [12]. The two-layer coating was able to protect the probiotics both under storage and during simulated gastrointestinal conditions.

The layer-by-layer technique involves the alternative adsorption of positively and negatively charged materials on surfaces through the chemical electrostatic deposition technique. This technique produced a protective outer layer on a microencapsulated probiotic by immersing the capsule in a biopolymer solution. This layer coating process can be repeated several times until the desired number of layers or thickness is obtained. The strength of the multilayer-coated capsule can be enhanced by increasing the interaction intensity between the charged materials. This is made possible by modification of the pH, concentration, and ionic strength of the polymer solution [31,45,60].

This technique does not involve high operational costs, as only mild conditions, aqueous solutions, and naturally charged materials are used in the coating process. The thickness, permeability, strength, and morphology of the layers can be tailored depending on the desired application. However, the adhesion times of each layer are between 1 and 60 min, which is not instantaneous. This leads to a certain degree of aggregation of the capsules during the adhesion of the subsequent layer, reducing the available surface area for consecutive layer adhesions [53,69].

## *3.6. Co-Encapsulation*

Co-encapsulation is an encapsulation method that utilizes the synergistic effect of two or more bioactive substances that can positively influence each other to enhance the function/viability of the encapsulated substances. This technique has been used in drugs and bioactive components in pharmaceutical industries [62]. However, in recent years, considerable attention has been given to co-encapsulation processes in food industries. Co-encapsulation has been proven to be able to sustain and enhance the viability of probiotics [17,18,27,29,35,36,70].

Co-encapsulation of probiotics together with prebiotics has received attention from food researchers. The effect of co-encapsulation of probiotics with arrowroot starch for yogurt was investigated in the study of Samedi and Charles [71]. After being stored in ambient and refrigerated conditions for 90 days, the co-encapsulated probiotics had higher viability when compared to the free probiotics. The probiotics co-encapsulated with arrowroot starch with low digestibility and prebiotic potential were more resistant to the harsh conditions in the gastrointestinal tract and acidic conditions in yogurt. Furthermore, Zaeim et al. [72] investigated the protective role of polysaccharide matrix (inulin or resistant starch in calcium-alginate/chitosan microcapsules) on the co-encapsulated probiotics (*L. plantarum* ATCC 8014 and *B. animalis* subsp. *lactis*) under gastrointestinal conditions and storage at -18, 4, and 25 ◦C. The presence of inulin and resistant starch in the microcapsules improved the survivability of these probiotics. Shinde et al. [73] also demonstrated that co-extrusion encapsulation of probiotic *L. acidophilus* with apple skin polyphenol extract using an aqueous delivery system possessed >96% microencapsulation efficiency and improved viability under low pH conditions (pH 2, 37 ◦C, 120 min) and after 50 days refrigeration storage (4 ◦C) in milk. Overall, encapsulated probiotics with resistant starch had stronger resistance against gastrointestinal conditions compared to the ones with inulin. Resistant starch could prevent gastrointestinal acid from diffusing into the microcapsules by entrapping within the porous alginate matrix. As the carbon source, resistant starch could improve the survival of probiotics during storage and also enhance the colonization and proliferation of probiotics in the intestines [72].

Table 1 shows the main properties, advantages, and disadvantages of encapsulation techniques that can be applied in multilayer and co-encapsulation techniques of probiotics.

**Table 1.** Overview of common probiotic encapsulation techniques.



#### **Table 1.** *Cont.*

## **4. Biomaterials Utilized for Probiotics Encapsulation**

To be an effective encapsulation material (core or wall material), the biomaterial used must be able to protect the encapsulated probiotics along the gastrointestinal tract until reaching the target site (small intestine/large intestine), where they can exert their health-promoting effects. The encapsulation material should only release the encapsulated probiotics when it is exposed and triggered by certain environmental conditions (such as temperature, pH, and enzyme activity). In other words, the capsules containing probiotics should remain protected inside the encapsulation material during the passage through the stomach and only decompose after reaching the target site to release the probiotics. The commonly used biomaterials in probiotic encapsulation include carbohydrates, proteins, and lipids, which will be discussed in detail in the coming subsections. Their specific advantages and limitations in probiotic encapsulation are also summarized in Table 2.

#### **Table 2.** Common biomaterials for encapsulating probiotics.



#### **Table 2.** *Cont.*


#### **Table 2.** *Cont.*

## *4.1. Carbohydrate Polymers*

## 4.1.1. Alginate

Among the carbohydrate polymers used, the most common biomaterial is alginate. Alginate can be produced by various brown seaweeds (*Laminaria digitata*, *Laminaria hyperborea*, *Laminaria japonica*, *Macrocystis pyrifera*, and *Ascophyllum nodosum*) and two genera of bacteria (*Pseudomonas* and *Azotobacter*), making it abundant and comparatively low in cost [45]. Alginate is the preferred biomaterial in probiotic encapsulation owing to its nonharmful nature, ease in producing strong beads, and being promptly accessible. Alginate has good gelling, balancing out, and thickening properties, and is easy to manipulate, biocompatible, and biodegradable [1,40,45,79]. Alginate is a pH-responsive polymer that is stable at lower pH and unstable in higher pH conditions which is beneficial in customizing release profiles. During the delivery, alginate beads tend to shrink in low acidic gastric environments. Hence, it prevents the release of the encapsulated probiotics from the beads. Once the beads reach the small intestine with alkaline conditions, the alginate transforms into a soluble alginic acid layer. Subsequently, they swell and release the encapsulated probiotics [75]. Unfortunately, alginate is sensitive to heat treatment, porous, unstable, and has poor barrier properties because of its high molecular mobility and weak interaction between the molecular chains [1,45]. The weakness of alginate can be overcome through a crosslinking reaction with divalent cations or co-encapsulation with starch or by coating the alginate capsules with an extra layer (multilayer technique) made of a different type of biomaterial [92]. The ionic crosslinking of alginate chains with calcium cations could result in a strong gel structure. The presence of calcium cations could also disrupt the water coordination of the alginate network [79]. The synergistic effects of alginate and starch of the alginate capsules could protect the entrapped probiotics [45]. With the interaction of the negatively charged carboxylic groups of alginate with positively charged amine groups chitosan, stronger, ordered three-dimensional gel networks can be produced. The resulting capsules also have smoother surfaces with decreased water permeability [67].

#### 4.1.2. Chitosan

Chitosan originates from chitin which is naturally synthesized by algae and the shell waste of crab, shrimp, and crawfish [45]. Chitosan-based hydrogels have been extensively employed to deliver probiotics owing to their unique cationic character, non-toxicity, high biocompatibility, biodegradability, bio-adhesiveness, inexpensive nature, antimicrobial, and antifungal properties [67,75,93]. Chitosan also has high tolerance against the deteriorative effects of calcium chelating and anti-gelling agents [67]. However, chitosan is a pH-sensitive material that tends to degrade in low pH conditions and is water-insoluble at pH > 5.4. Therefore, it is less effective in the delivery of probiotics to the gut [75]. Moreover, using chitosan as a polymer for entrapping live lactic acid bacteria (LAB) could exhibit inhibitory effects on the LAB [1]. Therefore, it is commonly applied as a coating or shell rather than a capsule matrix. Chitosan has been extensively used in combination with other biomaterials, including alginate, starch, whey protein isolate, and xanthan gum [1,45]. Chitosan coating could enhance the porosity of alginate beads, thus, reducing leakage of encapsulated bacteria and improving the pH stability of beads [67]. Chitosan coating increased the release rate of probiotics from alginate/starch beads and enhanced the survivability of probiotics in low pH conditions [94]. The chitosan coating on alginate/whey protein

isolates beads increased the resistance to thermal, storage, and simulated gastrointestinal environment [95]. In addition, the heat resistance of microcrystalline cellulose/Xanthan gum beads has also been enhanced by chitosan coating [32]. Encapsulation of probiotics using microcrystalline cellulose powder and alginate, or Xanthan gum followed by coating with chitosan (0.5%) as the outermost layer is effective in protecting probiotics (*L. Sporogenes*) against the baking process (90 ◦C for 15 min) in bread making.

## 4.1.3. Gums

Xanthan gum has been proven as an excellent embodiment in conferring protection against harsh gastrointestinal conditions and elevated temperatures (up to 90 ◦C for 5 s) to probiotics. It is an exopolysaccharide obtained through fermentation by *Xanthomonas campestris* from agro-industrial wastes [79]. Xanthan gum possesses an anionic character, is non-toxic, biodegradable, biocompatible, highly soluble in cold and hot water, and has excellent gelling properties. Furthermore, it has excellent heat and acid stability and is highly resistant to gastrointestinal digestion and enzymatic decomposition [41,82]. The study of Thang et al. [33] indicated the protective effect of Xanthan gum on the viability of *L. acidophilus* incorporated in bread under simulated gastric and intestinal conditions compared to using alginate alone. Xanthan gum has a negative charge structure that could bind to H<sup>+</sup> ions and minimize the effect of an acidic condition on the probiotics. Unfortunately, it has some limitations. It is susceptible to microbial contamination, unstable viscosity, and uncontrollable hydration rate, as well as producing gels with poor shear resistance, mechanical strength, and thermal properties when used solely [41,83]. Therefore, to enhance the coating properties of Xanthan gum in probiotic encapsulation, it is combined and used with other biomaterials, including alginate, chitosan, gellan, and β-cyclodextrin [41]. In contrast to alginate beads, the combination of xanthan and gellan gums produces capsules with higher resistance toward acid conditions [1,41].

Gellan gum is a microbial polysaccharide, industrially produced through fermentation by *Sphingomonas elodea* and *Pseudomonas elodea*. It is available in two forms, low acyl gellan gum (deacylated; Kelcogel) and high acyl gellan gum (acylated; Gelrite). Upon cooling, gellan gum with lower acyl contents (gel setting temperature: 40 ◦C) forms a more rigid and brittle gel, whereas gellan gum with higher acyl contents (gel setting temperature: 65 ◦C) tends to produce gels with a softer and more flexible texture. In probiotic encapsulation, low acyl gellan gum is commonly used [41]. In general, gellan gum is negatively charged, non-toxic, biocompatible, biodegradable, relatively cheap, and water-soluble. Gellan gum has high resistance against heat, enzymatic degradation, acidic environments, and swells in alkaline conditions, allowing it to be suitable as a controlled release polymer. However, the gel formed by gellan gum is considerably poor in mechanical strength and unstable in physiological conditions. The high gel-setting temperature (80–90 ◦C) of gellan gum also causes heat injuries to probiotics. Usually, it is used in combination with other biomaterials such as gelatin, sodium caseinate, and alginate in probiotic encapsulation [44,84]. Gellan gum has been used to increase the thermal stability of probiotics in a study on probiotic bread [32]. Results demonstrated that the gellan gum (1.5%) coating layer increased the survivability of *L. Sporogenes* encapsulated in alginate beads 24 h after baking.

Gum Arabic (or gum acacia) is another common biomaterial used in probiotic encapsulation. It is an arabinogalactan polysaccharide-protein anionic complex that provides surface activity, foaming abilities, and emulsifying characteristics. Gum Arabic possesses acid stability, high water solubility, and low viscosity even at a high concentration [41]. Gum Arabic has high water solubility, relatively low viscosity, and good film-forming and emulsifying properties, which reduces the hygroscopicity and degree of caking of the obtained powder. At the same time, it can prevent complete dehydration of probiotics during the drying process and storage. Hence, gum Arabic has been extensively used in spray-drying [78]. For instance, gum Arabic provided good protection to *L. acidophilus* from spray-drying damage. The viability of *L. acidophilus* encapsulated with gum Arabic was reduced by only 1 log CFU/g after being treated with spray-drying [70]. Gum Arabic tends

to produce microcapsules with irregular shapes and rough surfaces, which can reduce the ability to retain the probiotics. Gum Arabic is also a comparatively expensive ingredient because of frequent supply shortages. It shows partial protection against oxygen. Hence, it is used together with other biomaterials such as maltodextrin, gelatin, and whey protein isolates [41,84]. The use of gum Arabic in combination with maltodextrin (survivability of probiotics: 71.0%) was proven to provide better protection to probiotics during storage (10 weeks) than gum Arabic (35.3%) or maltodextrin (30.2%) alone [69]. Gum Arabic has also been used with β-cyclodextrin to produce the spray-dried probiotics (*S. boulardii*, *L. acidophilus*, and *B. bifidum*) in the production of probiotics-enriched cream-filled cake, marmalade-filled cake, and chocolate coated cake [31].

## 4.1.4. Starch

Starches have received great attention in the probiotic encapsulation process because they are generally recognized as safe, abundant, inexpensive, non-allergenic, able to produce a gel with a strong and flexible structure, transparent, colorless, flavorless, and odorless gel that is semi-permeable to water, carbon dioxide, and oxygen [76]. Probiotics can survive in gastrointestinal and colon environments when embodied in the starch granules [40]. Moreover, the utilization of starch with combinations of biomaterials, such as alginate and chitosan, was reported to protect the probiotics [41]. Chemically modified starches (e.g., succinated, cross-linked, substituted, oxidized, and acid-treated) possess higher solubility and better mechanical properties and have also been used in probiotic encapsulation [41,76]. Starch, i.e., resistant starch, can also serve as a potential prebiotic since this type of starch cannot be digested in our small intestines. The prebiotic effects of resistant starch allow a higher release of the probiotics in the large intestine. The adherence of the probiotics is also higher with resistant starch due to its robustness and resilience to environmental stresses [1]. Starch adhesion increases the initial cell load of probiotics and improves the targeted delivery of probiotics. However, starch often exhibits high viscosity in solution. Thus, it negatively affects the efficiency of encapsulation [67]. The starch viscosity can be adjusted through starch modifications.

## 4.1.5. Cellulose and Cellulose Derivatives

Cellulose is the most abundant biopolymer found in nature. Cellulose and its derivatives have been extensively used in probiotic encapsulation owing to their non-toxic character, biocompatibility, tunable surface properties, and pH-controlled release ability. Cellulose is insoluble at pH ≤ 5 but soluble at pH ≥ 6. Hence, it is effective in delivering probiotics to the colon. Common celluloses used in probiotic encapsulation are carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, and microcrystalline cellulose. An optimized delivery with a sustained and slow release of probiotics to the targeted region (intestine tracts) has been developed on cellulose-based gel beads [77]. Youssef et al. [94] reported that the viability of *L. salivarius* subsp. *salivarius* CGMCC No. 1.1881 encapsulated in alginate and coated with cellulose (carboxymethyl cellulose) was higher than the probiotics encapsulated in alginate without coating under thermal treatment, storage, and simulated gastrointestinal conditions. The main limitation of celluloses is that they cannot form gel beads using the extrusion technique [77].

## 4.1.6. Maltodextrin

Maltodextrin is one of the most common wall materials used in spray-drying to encapsulate probiotics. It is a starch hydrolysate produced from any starch via partial acidic or enzymatic hydrolysis. Maltodextrin is abundant, inexpensive, non-toxic, bland in taste, possesses low hygroscopicity, shows excellent thermal stability, has high water solubility, and low viscosity, even when at a high solid content [41,78]. These properties prevent particle agglomeration and contribute to the easy spray-drying of maltodextrin. It also possesses moderate prebiotic properties and is beneficial in probiotic encapsulation. However, maltodextrin has a weak emulsifying capacity. Hence, it shows a low encapsulation efficiency. In this regard, maltodextrin is often used together with other biomaterials such as gum Arabic and sodium caseinate [41]. Thang et al. [33] reported that the survivability of *L. acidophilus* during the bread-baking process was increased through the addition of maltodextrin in the encapsulation matrix. A higher protective effect was observed when maltodextrin was used with Xanthan gum.

## 4.1.7. Carrageenans

Carrageenans are natural hydrophilic polymers extracted from red seaweeds (Rhodophyta). Among the three commercially available carrageenans (κ-, ι-, and λ-carrageenan), κ-carrageenan is the most widely used in the encapsulation of probiotics [1]. This is due to the thermosensitive and thermoreversible characteristics of κ-carrageenan, making it a suitable material to deliver probiotics as the release can be controlled with temperature [79]. In general, encapsulation using κ-carrageenan involves the addition of probiotics to melted (at 80–90 ◦C) κ-carrageenan during the cooling period (at 40–50 ◦C). The encapsulation is completed when gelation occurs, i.e., when the reaction mixture is cooled to ambient temperature [1]. Nevertheless, the gels produced using κ-carrageenan are brittle and weak [41]. The properties of the formed gel can be enhanced by combining it with ι-carrageenan, locust bean gum, alginate, and carboxymethyl cellulose [41,79]. κ-carrageenan hydrogels have been used to deliver probiotics. It is observed to increase the viability of probiotics under gastrointestinal conditions and storage at refrigeration conditions (4 ◦C) and room temperature at 22 ◦C [79]. However, the rate of probiotic release from carrageenan-based hydrogels is much slower than from alginate-based hydrogels [41]. Carrageenan (κ-carrageenan) has also been used to enhance the viability of *B. bifidum* incorporated in grape juice [7]. The viability of *B. bifidum* encapsulated with carrageenan (7.09 log CFU/mL) was higher than the free probiotics (6.58 log CFU/mL) after being stored for 35 days.

## 4.1.8. Pectin

Pectin is a heteropolysaccharide that can be extracted from various kinds of fruits but commonly from the peels of citrus fruits. Pectin has been extensively used as a substitute for expensive biomaterials in the encapsulation of probiotics owing to its abundance, affordable price, anionic and non-toxic character, biocompatibility, biodegradability, bioadhesives, antimicrobial, and antiviral properties. Pectin possesses excellent gelling, thickening, and water-binding properties. In addition, it can form emulsions at low concentrations, making it suitable to be incorporated into spray-drying techniques. Pectin has been used with maltodextrins as probiotics (*L. casei* Shirota, *L. casei* Immunitas, and *L. acidophilus* Johnsonii) carrier in the production of probiotic enriched orange powder [24]. The combination of pectin and maltodextrins effectively enhanced the stability of probiotics during the spraydrying process. Pectin is resistant to gastric and intestinal enzymes but can be rapidly fermented by gut microbiota, thus facilitating the controlled release of probiotics in the gut. It is also an effective prebiotic that can enhance growth, increase acid tolerance, and improve the survival of encapsulated probiotics [75,80,81]. However, due to the high solubility of pectin in the aqueous medium, the bead produced by pectin shows limitations in its rate of diffusion and release of probiotics. Pectin beads have high porosity, low thermal stability, and mechanical strength. During the gelation of pectin, sucrose content was observed to increase. Therefore, pectin-based beads were not recommended for patients with diabetes [81].

#### *4.2. Protein*

In the past, proteins have been widely used as biomaterials in the encapsulation of probiotics. Potential plant-based proteins include maize (zein) and soy proteins, whereas animal-based proteins include gelatin, whey proteins, and milk proteins. In general, proteins possess an amphiphilic character, high emulsifying capacity, gel-forming ability, film formation capability, water solubility, biocompatibility, and biodegradability, allowing them to be excellent encapsulating materials. However, protein conformation and encapsulation

efficiency depend on the pH, ionic strength, and temperature. For instance, proteins are commonly used in combination with carbohydrate-based biomaterials. The main concern of using proteins as encapsulants is their allergenicity. Usually, plant-based proteins are less allergenic than animal-based proteins. The applications of animal-based proteins have also been limited by vegetarian and kosher trends, lactose intolerance, and other dietary restrictions [41].

## 4.2.1. Plant-Based Proteins

#### Zein Proteins

Zein is the major protein of maize. Owing to its amino acid residues with polar and non-polar side chains, zein exhibits an amphiphilic character. It is suitable for use as a biomaterial for encapsulations and delivery of water-insoluble probiotics. In addition, zein has high resistance against gastric juice. Hence, it can extend the release of probiotics in the small intestines. Despite its high surface hydrophobicity, zein is highly unstable and tends to aggregate in aqueous solutions. For instance, zein beads are commonly coated with a layer of emulsifiers such as sodium caseinate and Tween 20 or ionic polysaccharides such as alginate and pectin [89].

Riaz et al. [96] used zein protein-coated alginate microbeads to encapsulate *B. bifidum*. The probiotics encapsulated in zein protein (1, 3, 5, 7, and 9% (*w*/*v*))-coated alginate microbeads were higher in viability compared to those encapsulated in alginate microbeads (105 log CFU/g) and free cells (103 log CFU/g) after being stored for 32 days at 4 ◦C. Zein protein coating also enhanced the resistance of encapsulated *B. bifidum* against the harsh conditions in gastrointestinal transit. Zein protein (5% (*w*/*v*)) coating was also observed to increase the viability of carboxymethyl cellulose-coated *L. plantarum* 299v in apple slices under simulated gastrointestinal conditions [12].

## Soy Proteins

To date, the utilization of soy protein in the encapsulation of probiotics is rare. Protein isolated from soybean is a potential probiotic encapsulation biomaterial owing to its high nutritional value, less allergenic nature, and good emulsifying, absorbing, and film-forming properties [41,89]. Soy proteins also possess high resistance against gastric juice. Therefore, they are efficient in delivering and controlling the release of probiotics to the gut. Nevertheless, heat-induced gel formation of soy proteins can affect the viability of heat-sensitive probiotics. Heat treatment could also cause protein denaturation, resulting in loss of functionality [90].

Soy protein isolates have been used with gum Arabic to prepare nano-emulsion to encapsulate *L. delbreuckii* subsp. *Bulgaricus* [50]. The presence of soy protein isolates in the emulsion can increase the stability and enhance the survival rate of probiotics during storage (at 27 ◦C for 40 days). In another study, soy protein isolates (20% (*w*/*v*)) were employed with sodium alginate (4% (*w*/*v*)) to encapsulate *L. plantarum* using the extrusion technique. The inclusion of soy protein isolates increased the thermal resistance of *L. plantarum*. The viability of *encapsulated L. plantarum* (a slight decrease from 9.10 log to 8.11 log CFU/mL) in mango juice remained high after the pasteurization process [21].

## 4.2.2. Animal-Based Proteins Gelatin

Gelatin is a heterogeneous mixture of water-soluble proteins that can be obtained through the partial hydrolysis of collagen derived from various sources, e.g., bones, skin, scales, and connective tissues of animals [85]. When dissolved in hot water, gelatin forms a thermoreversible gel, which has been used (both on its own and with other biomaterials) to encapsulate probiotics [1,85].

Gelatin can combine with many different polysaccharides, making it one of the most studied proteins in probiotic encapsulation [1,41]. Amphoteric gelatin can be used with anionic polysaccharides (synergistic effects) to form capsules that are tolerant against cracking and breaking. The linear structure of gelatin also provides a better oxygen barrier when compared to globular proteins [1].

## Whey Proteins

Whey proteins are a complex mixture of globular proteins isolated from whey, which refers to the liquid part of milk (by-product) that separates during the cheese-making process. Whey is mainly constituted of β-lactoglobulin (85%), α-lactalbumin (10%), and bovine serum albumin (5%). β-lactoglobulin, the major protein in whey, is rich in rigid β-sheet structure and two disulfide bonds. These two unique features of β-lactoglobulin provide whey a high resistance and stability against pepsin digestion, making whey protein a suitable encapsulation material for the controlled release of probiotics [86]. Previously, whey proteins have been found to increase the resistance of probiotics against gastrointestinal conditions for up to 3 h [45,88]. Whey proteins, including whey protein concentrates (35−85% protein) and whey protein isolates (>95% protein), have been used in probiotic food products as encapsulating materials [41]. Whey proteins are a suitable medium to preserve and deliver probiotics owing to their high nutritional composition (containing soluble milk proteins and lactose). Whey proteins also possess amphoteric character, good gelation properties, thermal stability, hydration, and emulsification properties (pre-treated by heat-induced denaturation). Hence, they can interact, entrap, and protect probiotics components [87]. In probiotic encapsulation, whey proteins have been used as wall materials together with gum Arabic, maltodextrin, and pectin. The synergistic effects between whey proteins and polysaccharides have been reported to enhance the encapsulation efficiency of whey proteins [86].

#### Caseins

Caseins are a promising encapsulating material for probiotics owing to their structural and physicochemical properties. Caseins have excellent gelation properties and can form gels under mild conditions through different techniques, including extrusion, emulsification, spray-drying, and acid- and enzyme-induced gelation. As one of the protein components in milk, casein accounts for almost 80% of milk's total protein content. Moderate viscosities of caseins have contributed to easy dispersion of the probiotics, producing gel beads with high density and better protection for the encapsulated probiotics. The strong amphiphilic character allows caseins to encapsulate both hydrophilic and hydrophobic probiotics. Caseins can also produce gel beads of varying sizes (ranging from 1 to 1000 μm). In probiotic encapsulation, sodium caseinate is most used, owing to its excellent emulsifying properties and high resistance to thermal denaturation [41,88,89].

## *4.3. Lipids*

Lipid matrices, such as fatty acids, diglycerides, monoglycerides, vegetable-based oils, waxes, and resins, are commonly used to encapsulate hydrophilic probiotics [81,82]. Lipidbased biomaterials are naturally low in polarity, exhibit excellent water barrier properties, and are thermally stable [76,92]. However, lipid-based biomaterials have weak mechanical properties and are chemically unstable. Therefore, lipids are often combined with other biomaterials, such as polysaccharides or proteins, to increase their performances in probiotic encapsulation [76]. In addition, when used with other biomaterials, capsules with low gas migration can be produced [41,76]. Compared to free *L. casei* and *B. pseudolongum*, lipid encapsulated probiotics were observed to have higher viability under simulated intestinal conditions [41]. However, this improvement was not observed during storage. In addition, lipid-based biomaterials were reported to have adverse effects on the overall sensory characteristics of the food product carrying the probiotics owing to lipid oxidation [41,76].

## **5. Application of Probiotics Encapsulation in Non-Dairy-Based Food and Beverage Products**

The growing demand for non-dairy probiotic food products has encouraged scientists and researchers to explore more new non-dairy food matrices (Table 3). Recent studies have proved that non-dairy food matrices (known to be free of lactose, dairy allergens, and cholesterol and rich in nutrients) are promising vehicles for probiotic delivery. Furthermore, the probiotics were also observed to adapt well to encapsulation using non-dairy food matrices owing to their richness in nutrients. However, researchers still face some challenges, such as the maintenance of probiotic viability and sensory properties of probiotic food products [2,9]. For instance, the composition, pH value, and storage condition of the nondairy food substrate could negatively affect the viability of inoculated probiotics. Under certain conditions, the metabolic compounds produced through the interaction between the probiotics and food matrices could negatively affect the sensory qualities of non-dairy food products. While probiotics do not usually replicate in non-dairy matrices, it is necessary to keep the viability of probiotics at an adequate level. In addition, components such as carbohydrates, proteins, and flavoring agents in the food matrix could also negatively affect the viability of probiotics. Encapsulated probiotics with bigger particle sizes were also reported to be adverse to the mouthfeel sensation.

**Table 3.** Examples of recent application of probiotics encapsulation in non-dairy-based products.



#### **Table 3.** *Cont.*


#### **Table 3.** *Cont.*


#### **Table 3.** *Cont.*

## *5.1. Fruit and Vegetable-Based*

In contrast to dairy products, fruit and vegetable juices do not contain allergens, lactose, and cholesterol. In addition, the main macronutrients in fruit and vegetable juices are carbohydrates and dietary fibers, and they are rich in vitamins, minerals, polyphenols, phytochemicals, and antioxidants. In the sensory aspect, fruit and vegetable juices are refreshing and usually do not have undesirable tastes and flavors. Therefore, fruit and vegetable juices have been recognized as promising carriers for probiotics for all age and economic groups [2,9].

Several factors could limit the survivability of probiotics in fruit and vegetable juices, including the type of probiotic strain used, the conditions of medium (e.g., pH, water activity, oxygen stress, presence of antimicrobial compounds, dyes, flavors, and preservatives), as well as the process of juice production (e.g., pasteurization process, storage temperature, type of packaging material used, and food handling practices) [9]. Among the factors, the pH condition of the medium used has the most effect on the viability of probiotics. Fruit juices naturally have a low pH value, while vegetable juices are generally less acidic. It has been reported that *Lactobacilli* can resist and survive in pH conditions ranging from 3.7 to 4.3; however, *Bifidobacteria* are less acid tolerant. Recently, encapsulated probiotics (*B. animalis*, *B. bifidum*, *E. faecium*, *L. acidophilus*, *L. casei*, *L. fermentum*, *L. lactis*, *L. plantarum*, *L. sphaericus*, and *S. boulardii*) were incorporated into fruit and vegetable juices, such as carrot, cherry, grape, mandarin fruit, mango, orange, passion fruit, pineapple, raspberry, Sohiong, sugar cane, and tomato juices [7,13–18,20–22].

Sour cherry juice has an approximate pH value of 3.5, rendering it an unsuitable medium for delivering probiotics. Encapsulation (technique: extrusion, material: sodium alginate) increased the viability of *E. faecium* in sour cherry juice during storage (from 2.18 to 5.39 log CFU/mL, 4 ◦C for 60 days; from 4.30 to 6.25 log CFU/mL, 25 ◦C for 21 days) and its tolerance against heat, acid, and digestion treatments [13]. Although alginate is the most used biomaterial in protecting probiotics, it is susceptible to low acid conditions. Lowacidic conditions change the particle shape of alginate beads, resulting in adverse effects on the release rate. In a recent study [17], Persian Gum was used with alginate and prebiotics (FOS and inulin) to encapsulate *L. lactis* ABRIINW-N19 before being added to orange juice. Among the formulations tested in the study, alginate–Persian Gum + 2% inulin was the best as it contributed the highest encapsulation efficiency and best protection for the probiotics against harsh gastrointestinal conditions. Alginate–Persian Gum + 2% inulinencapsulated *L. lactis* also showed the highest viability during the storage period. In addition, it exhibited the best cell release activity and buffering ability in orange juice. The application of evolved extrusion technique (vibrating nozzle method) to encapsulate *L. casei* DSM 20011 was demonstrated by Olivares et al. [18]. However, the vibrating nozzle method and biomaterial used (alginate) were reported to be insufficient in protecting the probiotics as the acidic conditions could still negatively affect the viability of *L. casei* even when encapsulated. According to Olivares et al. [18], the addition of antimicrobial compounds, such as anthocyanins, can affect the viability of probiotics. Praepanitchai et al. [21] also utilized the extrusion technique to encapsulate *L. plantarum* in the developing probiotics-enriched mango juice. Soy protein isolate (20% (*w*/*v*)) used in encapsulation increased the thermal resistance of *L. plantarum* in mango juice, i.e., a slight decrease in the viability of *encapsulated L. plantarum* was observed after the pasteurization.

Generally, the pH value of grape juice ranges between 3.0 and 4.0. Using the emulsion technique, Mokhtari et al. [15] and Afzaal et al. [7] showed that the survivability of probiotics in grape juice can be improved. Both researchers encapsulated their probiotics in alginate beads, while Afzaal et al. [7] also encapsulated *B. bifidum* with κ-carrageenan. Similar findings were observed in both studies, whereby the viability of encapsulated probiotics in grape juice is higher than those of non-encapsulated. The survivability of *L. acidophilus* and *B. bifidum* with encapsulation (8.67 and 8.27 log CFU/mL, respectively) was higher than free probiotics (7.57 and 7.53 log CFU/mL, respectively) after being kept refrigerated (4 ◦C) for up to 2 months [15]. The survivability of *B. Bifidum* was enhanced

from 6.58 to 8.51 log CFU/mL (encapsulated with sodium alginate) and 7.09 log CFU/mL (encapsulated with κ-carrageenan) after 35 days of storage [7]. The encapsulated probiotics were also observed to have stronger resistance against simulated GI conditions when compared to free probiotics [7].

Similarly, Naga Sivudu et al. [16] also utilized the emulsion technique to encapsulate probiotics (*L. plantarum*, *L. fermentum*, *L. casei*, *L. sphaericus*, and *S. boulardii*) in juices (tomato and carrot juices), but with an additional of chitosan coating at the outer layer of alginate capsule. Although encapsulated probiotics had higher viability than free probiotics during refrigerated storage (4 ◦C for 5–6 weeks), the beads negatively influenced the sensory quality of the juice. The vegetable juices with encapsulated probiotics were reported as hard to swallow and highly turbid.

Sugarcane juice is a relatively new matrix used to deliver probiotics. In the study carried out by Holkem et al. [14], *B. animalis* was co-encapsulated with concentrated whey protein, gum Arabic, and proanthocyanidin-rich cinnamon extract through a complex coacervation technique. The encapsulation showed an increment in the probiotics' survivability and retention of the phenolic and proanthocyanidin compounds in the sugarcane juice. However, encapsulated probiotics and proanthocyanidin-rich cinnamon extract altered the viscosity of sugarcane juice. This is adverse to the sensory properties of the juice. The complex coacervation technique has also been used by Silva et al. [22] in probiotic orange and apple juices. The encapsulated *L. acidophilus* LA-02 incorporated in fruit juices survived throughout the refrigerated storage (4 ◦C for 63 days).

By using the spray-drying technique, Vivek et al. [20], Gervasi et al. [24], and Santos Monteiro et al. [19] successfully obtained fruit powder rich in probiotics. Encapsulation with magnesium carbonate and maltodextrin, the viability of *L. plantarum* (6.12 log CFU/g) in Sohiong juice powder was maintained for 36 days without refrigeration [19]. In the study of Gervasi et al. [24], *L. casei* Shirota, *L. casei* Immunitas, and *L. acidophilus* Johnsonii were encapsulated by using pectin and maltodextrin before spray-drying together with orange juice. The combination of pectin and maltodextrin was reported to enhance the stability of probiotics during the spray-drying process. On the other hand, Santos Monteiro et al. [19] claimed that a blend of gelatin and maltodextrin retained the viability of *L. reuteri* and phenolic compounds in passion fruit pulp against harsh conditions of the spray-drying process. In another study [23], freeze-drying and spray-drying were used to encapsulate *E. faecalis* incorporated in carrot juice using gum Arabic and maltodextrin as coating materials. The results showed that freeze-drying exerted fewer heat injuries on the probiotics than those spray-dried. Massounga Bora et al. [25] also used freezedrying to encapsulate probiotics (*L. acidophilus* and *L. casei*), using whey protein isolate and fructooligosaccharides as wall material, in the production of banana powder. Freezedried probiotics were observed to possess higher survivability under storage (4 ◦C for 30 days) and simulated gastrointestinal conditions than free probiotics. Probiotics (*L. plantarum* TISTR 2075) enriched carrot tablets were developed using the fluidized bed drying technique [26]. The results showed that the encapsulated probiotics in the tablets were more resistant to heat and digestion treatments when compared to the free probiotics.

Fruit pieces are also potential vehicles to deliver probiotics. In a recent study by Ester et al. [10], *L. salivarius* was encapsulated in alginate beads through the emulsion technique before adding to mandarin juice. The probiotic-supplemented mandarin juice was then used to incorporate *L. salivarius* into apple discs. The probiotics-impregnated apple discs were then dried and stored for 30 days. From the study, the encapsulated *L. salivarius* was found to have higher viability than free cells, indicating that encapsulation had improved the heat resistance properties of the probiotics. The encapsulation also proved to exert stronger resistance onto the probiotics against simulated gastrointestinal conditions. Wong et al. [12] also encapsulated probiotics (*L. plantarum* 299v) in apple slices. The *L. plantarum* 299v was coated with carboxymethyl cellulose followed by zein protein, and the coatings were reported to increase the resistance of probiotics under simulated gastrointestinal conditions. In another study, Galvão et al. [11] utilized a fluidized bed drying technique to dry and coat apple cubes with a mixture of hydroxyethyl cellulose and polyethylene glycol containing *B*. *coagulans*. The encapsulation was able to preserve the viability of probiotics in the dried apple snacks throughout the storage period.

Nowadays, non-edible parts of fruits have received much attention from researchers due to their abundance of bioactive compounds and promising functional properties. Recently, a powdered premix was developed using grape pomace, pomegranate, beetroot peel extract powders, and *L. casei* 431 co-encapsulated in quince seed gum-alginate hydrogel beads. Encapsulation increased the survival rate of *L. casei* throughout the freeze-drying process, from 42.16 (free cells) to 86.40% (normal encapsulation without the inclusion of prebiotic) and 87.56% (co-encapsulation with prebiotic). Quince seed gum–alginate hydrogel beads showed high encapsulation efficiency of 95.20% and maintained the viability of *L. casei* for up to 2 months [37].

## *5.2. Other Non-Dairy Based Products*

In addition to fruit and vegetable juices, tea and sap beverages have also been used as vehicles to deliver probiotics. Green tea is rich in polyphenols and was found to have various health-promoting effects. The presence of polyphenols has been reported to be able to improve the survival of oxygen-sensitive probiotics in aqueous solutions [73,97]. During storage, fermentation by the probiotics can occur, affecting the sensory acceptability of green tea. Furthermore, the polyphenols in green tea can also be adversely impacted, leading to the loss of its antioxidant activity. To address these adverse effects, Hernández-Barrueta et al. [28] encapsulated *L. rhamnosus* in a matrix of whey protein isolate in combination with modified huauzontle starch by spray-drying before incorporating it into green tea. After refrigerated storage (4 ◦C for 23 days), the green tea displayed high viability of probiotics (7 log CFU/mL). There was also no evidence of the occurrence of fermentation and insignificant variation in the antioxidant and polyphenolic contents of green tea.

In another work by Yee et al. [29], *L. acidophilus* NCFM (L-NCFM) was encapsulated in beads prepared using locust bean gum with and without mannitol (prebiotic) to develop a mulberry tea fortified with probiotics. Findings from the study revealed that L-NCFM encapsulated with the presence of mannitol showed the highest survivability (78.89%) and viable count (6.80 log CFU/mL) in the tea after a month of storage at 4 and 25 ◦C, respectively. Higher survivability was also observed in co-encapsulated L-NCFM under simulated gastrointestinal conditions compared to free and regular encapsulated (extrusion without prebiotic) probiotics. Similarly, using a co-encapsulation technique, Srisuk et al. [27] successfully introduced *L. acidophilus* TISTR 2365 into a sweet fermented rice sap beverage. During the encapsulation of probiotics into alginate beads, egg and fruiting bodies of bamboo mushrooms were added as prebiotics. The incorporation of an egg of bamboo mushroom at 3% was observed to increase the survival of *L. acidophilus* in the beverage most efficiently. The total phenolic contents and DPPH radical scavenging activities were also increased with the addition of the prebiotic.

Bakery products are recognized as staple foods worldwide, commonly consumed as breakfast, afternoon tea, and even evening snacks. However, bakery products are usually perceived as unhealthy as they contain high amounts of simple sugars and fats while being low in dietary fiber [98]. Hence, attempts have been made to improve the negative perception of bakery products, including incorporating probiotics into bakery products. Under typical probiotic incorporation into bakery products, whereby probiotics were added to the dough, a significant loss of viable probiotics in the bakery products is inevitable as these probiotics were killed by the high temperature used during baking. Although the loss of viability can be minimized by incorporating the probiotics directly into the cream filling or spreading them on the surface of the baked bakery product, not all bakery products are cream-filled. Arslan-Tontul et al. [31] investigated using single- and double-layered coated capsules to protect *S. boulardii*, *L. acidophilus*, and *B. bifidum* in cake. Double-layered encapsulation was found able to preserve the probiotics during the baking process. In a recent study, Mirzamani et al. [32] used an encapsulation method (fluidized bed drying) to

protect the *L. Sporogenes* in bread production. The encapsulated *L. sporogenes* in alginate (1%) capsules were observed to tolerate the simulated gastric acid condition. Incorporating chitosan (0.5%) into the outer layer increased the ability of probiotics to withstand heat. The highest survivability 24 h after baking was observed in encapsulated *L. sporogenes* with an outer layer coated with 1.5% gellan. In another study by Thang et al. [33], probiotics were incorporated into bread. It was reported that the survivability of *L. acidophilus* during the bread baking process was enhanced through the addition of maltodextrin and Xanthan gum in the encapsulation matrix.

Mayonnaise is used as an adjunct on salads, vegetables, and sandwiches. The high fat and high water activity of mayonnaise make mayonnaise a suitable carrier for probiotics in the human gut. In the study by Bigdelian and Razavi [35], *L. casei* Lc-01 and *L. acidophilus* La5 were added into mayonnaise in free and encapsulated forms (with and without prebiotic). Both *L. casei* and *L. acidophilus* encapsulated with high amylose maize starch (7.204 and 8.45 log CFU/mL) had higher viability than those without prebiotic added (7.1 and 7.94 log CFU/mL) and free cell (6.23 and 6.039 log CFU/mL) after refrigerated storage (4 ◦C for 91 days). Co-encapsulated probiotic cells had higher viability in mayonnaise throughout the storage than normal encapsulated (extrusion without prebiotic) probiotics. In addition, fewer chemical changes were observed in the mayonnaise sample supplemented with co-encapsulated probiotics.

Confectionery products are food products with minimal nutritional value and high sugar content. Over the years, the popularity of confectionery products has been on the rise among children. In this case, attempts have been carried out to incorporate probiotics into confectionery products, hoping to bring health benefits to consumers, especially children. Among the confectionery products, jelly and chewing gum are extensively consumed by all age groups. While high thermal treatments and low acidic conditions are unavoidable in producing jelly, Wulandari et al. [30] managed to maintain the viability of *L. plantarum* Mar8 (9 log CFU/mL) in black grass jelly for 14 days during refrigerated storage (4 ◦C) through microencapsulation using carrageenan. Alternately, a combination of inulin and lecithin was used as prebiotic sources with wall material alginate to co-encapsulate probiotics in the preparation of synbiotic chewing gum [36]. The prebiotics in encapsulation retained the viability of the *L. reuteri* during storage (for 21 days) without affecting the sensory properties of the chewing gum. The viability of *L. reuteri* was also reported to increase with the concentration of inulin and lecithin.

## **6. Conclusions**

With the ongoing popular trend of vegetarianism and an increasing number of lactoseintolerant and dairy-allergic consumers, the development of non-dairy delivery systems without lactose, dairy allergens, and cholesterol for probiotics has shown tremendous growth in recent years. Nevertheless, the development of non-dairy delivery systems is quite challenging because the composition, pH value, and storage condition of the non-dairy food matrices could negatively affect the viability of inoculated probiotics. Although encapsulation has been widely reported to be effective in preserving the viability of probiotics during storage, manufacturing, and gastrointestinal digestion, the techniques and biomaterials used are greatly dependent on the probiotic strain, the food matrix, and the food preparation method. Therefore, it is crucial to select appropriate techniques and biomaterials for the encapsulation and delivery of probiotics. Based on cited studies, coencapsulation of probiotics with prebiotics was found to be most effective in preserving the viability of probiotics in non-dairy food matrices.

**Author Contributions:** Project administration, funding acquisition, investigation, supervision, W.Y.K.; writing-original draft, visualization, writing—review and editing, W.Y.K. and X.X.L.; writing—review and editing, T.-C.T., R.K. and B.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by a grant provided by Universiti Malaysia Sabah, SPLB scheme (Grant No.: SLB2228).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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