Introduction

Ras cheese is an Egyptian traditional hard cheese prepared with a mixture of cow and buffalo milk. It is similar to the Greek variety “kefalotyri” (Phelan et al., 1993). This type of cheese requires a long ripening period to develop the full aroma and texture of matured cheese (El-Hofi et al., 2010). Consumers make their choice of this kind of cheese mainly on the quality and flavor characteristics, so it is a very important feature of cheese (El-Sayed et al., 2020). The flavor of ripened cheeses is principally affected by lipolysis and proteolysis of cheese curd, which are related to endogenous milk enzymes, microbial enzymes, and enzymes added for technological purposes (Crow et al., 1993). Recently, the Egyptian Organization for Standardization and Quality Control (ES, 1–1007: 2005) stipulated that Ras cheese should be produced from pasteurized milk or milk treated by heat equivalent to pasteurization. Also, it should be free from pathogenic microorganisms and their toxins, free from E. coli, the total coliform count should not exceed 10 CFU/g, and total mold and yeast counts should not exceed 100 CFU/g.

Herbs and spices have been used to fortify foods throughout history as preservatives, flavors, and therapeutic properties. Most spices are added to cheeses to impart unique flavors and enhance the microbiological quality of cheeses, and these cheeses are often considered specialty cheeses (El-Sayed & Youssef, 2019). Moreover, several studies have recommended herbs and spices for their beneficial and therapeutic effects on human health (Bhattacharyya et al., 2017; Bin et al., 2011).

The berries of (Pimenta dioica (L.) Merr.) belong to the family Myrtaceae. It is also called in English as allspice or pimento and takes its name from its aroma, which smells like a mix of cloves, ginger, nutmeg, and cinnamon. Allspice is used as a natural herbal remedy and has many health benefits such as aiding in digestion, anti-inflammatory qualities, boosting immunity, dental care, protecting heart health, and improving circulation. Moreover, allspice extracts have antibacterial and anesthetic properties, antioxidant capacity, anti-tumor activity, and utility in combating yeast and fungal infections (George & Joseph, 2013; Mandeel et al., 2003; Onwasigwe et al., 2017; Zhang, 2015). The concentrations of allspice extract to determine its antimicrobial activity against different pathogens were selected based on previous studies (Chaudhari et al., 2020; Khandelwal et al., 2012; Milenković et al., 2020; Murali et al., 2021) that found that the low concentration of the extract has an antimicrobial effect against different food spoilage and pathogenic strains.

Many technological challenges such as the microencapsulation process were carried out to increase dairy products enriched with herbs or spices in nanoform to increase their availability and efficiency. The microencapsulation process is characterized as a coating process of small particles or amalgamation of the compounds in a homogeneous matrix to protect these compounds from unfavorable ecological conditions and also to prevent interactions with other components (Fuchs et al., 2006; Favaro-Trindade et al. 2008). Moreover, microencapsulation is a suggested substitute to keep bioactive compounds correlated to antimicrobial activity. Also, the microencapsulation process was able to control the release of bioactive compounds from the microcapsules, masking the color, flavor, and odor of the compounds, preventing heat effect and oxidation when applied to food and dairy products (Ismail et al., 2020; El-shafei et al., 2018; Shrestha et al. 2017; Fareez et al., 2015).

There were numerous methods applied to produce microcapsules such as extrusion, conservation, and supercritical fluid precipitation (Farrag et al., 2018; Fayed et al., 2018). Spray drying and freeze-drying are the most used encapsulation methods that remove water and decrease water activity, avoiding the possibility of biological and chemical degradation, ensuring microbiological stability of products and lowering costs of transportation, as well as allowing immediate solubility of the product (Fioramonti et al., 2017; Gharsallaoui et al., 2007). For the encapsulation of sensitive bioactive substances, the freeze-drying method is an effective method. Rezvankhah et al. (2020) and Papoutsis et al. (2018) reported that the primary constituents in many plant extracts, known as phenols, are susceptible to elevated temperatures that ensue from the spray-drying process. Additionally, the obtained microcapsules are present in powder, which can be easily added, reconstituted, and dispersed well in the food matrix. The freeze-drying technique is simpler than other microencapsulation techniques.

This study aimed to identify and quantify the phenolic components found in allspice berries and to assess the antibacterial effectiveness of the extract nanoemulsion against foodborne pathogens. Using the freeze-drying procedure, the microcapsules containing the allspice berry extract nanoemulsion were prepared and characterized. The impact of enrichment with functional microcapsules on the physicochemical, microbiological, and sensory properties of Ras cheese was observed during 4 months of ripening.

Materials and Methods

Materials

Raw cow and buffalo milk (from the farm of the Faculty of Agriculture, Cairo University, Egypt) were used. Microbial rennet powder CHY-MAX (Chr. Hansen Holding A/S, Boege, 2970 Hoersholm, Denmark) was used for cheese production at the rate of 1 g/100 l. Whey protein concentrate (80%, from Agri-Mark, USA), guar gum (ScienceLab.com Chemicals Laboratory Equipment, USA), maltodextrin (Merck, Darmstadt, Germany), and HPLC-grade acetonitrile (J.T. Baker, NJ, USA) were utilized. Commercially dried allspice berries were purchased from an herbal market located in the government of Giza, Egypt. Nonionic surfactant Tween 80 (Sigma-Aldrich, Egypt) and Milli-Q (Millipore Corporation) water were used for all the experiments throughout the study. All chemical materials used in Ras cheese production were food grade.

Standards

Phenolic standards, namely, gallic acid, chlorogenic acid, catechin, methyl gallate, caffeic acid, syringic acid, pyrocatechol, ellagic acid, coumaric acid, rutin, vanillin, ferulic acid, naringenin, daidzein, quercetin, cinnamic acid, taxifolin, kaempferol, and hesperetin were purchased from Sigma-Aldrich (Egypt).

Microbial Strains

Pathogenic and spoilage strains used in antimicrobial activity tests include Salmonella typhimurium (14028s), Staphylococcus aureus (ATCC 6538), Bacillus cereus (B-3711), Listeria monoytogenes (598), Escherichia coli (ATCC 8739), Yersinia enterocolitica (ATCC 23715), Pseudomonas aeruginosa (ATCC 27853), Aspergillus niger (3858a), and Aspergillus flavus (B-3357). All strains were collected from the Dairy Department, National Research Centre. Starter cultures of Lactococcus lactis spp. lactis and Lactococcus lactis spp. cremoris were accessed from the Food Science Department, Faculty of Agriculture, Ain Shams University, Egypt.

Extraction Procedure of Dried Allspice Berries

An electric grinder was used to grind dried allspice berries to a fine powder. Fifty grams of powder was extracted with ethanol through maceration for 2 days at 500 ml and stirred at room temperature (Fig. 1). The resultant solution was filtered and dried under vacuum at 40 °C using a rotary evaporator. The obtained dry extract was converted to nanoemulsion form and produced as microcapsules to apply in the production of Ras cheese.

Fig. 1
figure 1

Flow sheet diagram for preparation of dried allspice berry extract

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Preparation of Allspice Fruit Extract Nanoemulsion

Allspice berry extract nanoemulsion was formulated using oil-in-water nanoemulsion through organic allspice extract (10%), and nonionic surfactant Tween 80 was desired because of its high hydrophilic-lipophilic balance (HLB) value that is auspicious for fabricating oil-in-water nanoemulsion as well as distilled water. Tween 80 has an HLB value of 15. At first, the emulsion was fabricated by the addition of distilled water to the organic phase comprising organic allspice extract and surfactant in ratio 1:3 (v/v) by a magnetic stirrer at 800 rpm for 10 h. The formed emulsion was exposed to ultrasonic emulsification with a 20 kHz Sonicator (Ultrasonics, USA) using the highest power output of 750 W. The sonicator rod was proportionally dipped into organic allspice extract, establishing a coarse emulsion. Furthermore, the sonicator probe produces disruptive forces that decrease the droplet diameter, changing coarse emulsion to nanoemulsion. The formulated allspice extract nanoemulsion was stored in a brown flask for additional investigations.

Microencapsulation Technique for Allspice Berry Extract Nanoemulsion

The coating material composition was prepared by dissolving in sterilized distilled water with the following materials: 15.4 g maltodextrin, 3.9 g whey protein concentrate, and 0.5 g guar gum according to Ogrodowska et al. (2017). All coating materials were blended well and stirred for 30 min to dissolve the mixture. The concentration of allspice extract nanoemulsion (10.6%, w/v) was blended with the coating material mixture by a high-pressure homogenizer (Ingenieurbüro CAT, Germany) at 9000 rpm for 120 s at 40 °C. Also, before freezing, the resultant emulsion was homogenized again at 9000 rpm for 1 min at 40 °C. The mixture was frozen at − 20 °C for 24 h and then freeze-dried using a lyophilizer (ALPHA 1–4 LSC). Freeze-drying was performed at a pressure of 0.12 mbar, and drying time reached 72 h at − 40 °C. After the freeze-drying process, the sample was ground gently by using a glass rod to transform it into a separated microcapsule form.

Microencapsulation Efficiency (ME %)

The microencapsulation efficiency for the allspice extract was measured as the total phenol content, which is considered the major component of the extract. The microencapsulation efficiency was evaluated by the Folin-Ciocalteu method according to Bae and Lee (2008) and Farrag et al. (2018). One gram of the obtained microcapsules was dissolved in 10 ml ethanol/acetic acid/water (50:8:42) and stirred well by vortex for 1 min. The dissolved microcapsules were filtrated by Whatman filter paper No. 1. The absorption was measured by using a UV/VIS spectrometer at 760 nm (model 2010, Cecil Instr. Ltd., Cambridge, UK) as color changes, which the Folin-Ciocalteu reagent was reduced by sodium carbonate in the presence of phenolic substances. For determining the surface phenolic content, 1 g of microcapsules was dispersed with 10 ml of ethanol and methanol mixture (1:1, v/v) for 1 min. The phenolic content of the surface was measured with the same method by Folin-Ciocalteu. Then, the microencapsulation efficiency (ME) was calculated using the following equation:

$$\mathrm{ME}\;\%=\frac{\mathrm{total}\;\mathrm{phenolic}\;\mathrm{content}-\mathrm{surface}\;\mathrm{phenolic}\;\mathrm{content}}{\mathrm{total}\;\mathrm{phenolic}\;\mathrm{content}}\times100$$

HPLC Conditions of Phenolic Compounds of Allspice Berry Extract

HPLC system (Agilent 1260) was separately carried out for the phenolic compounds using Eclipse C18 column (4.6 mm × 250 mm i.d., 5 μm). The injection volume was 10 μl for each of the sample solutions. The gradient elution was accomplished using water (solvent A) and 0.05% trifluoroacetic acid in acetonitrile (solvent B). The mobile phase flow rate was 1 ml/min. The program of the mobile phase is presented in Table 1. The column temperature was retained at 35 °C. The multi-wavelength detector was examined at 280 nm.

Table 1 The gradient of elution solvents
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Morphology of Nanoemulsion Droplets

Transmission electron microscopy (TEM, model: JEOL-JEM-1400) was done to determine the shape and morphology of the allspice extract nanoemulsion. To achieve the TEM investigations, one drop of the prepared nanoemulsion was negatively stained with phosphotungstic acid and was located on a copper grid.

FT-IR Investigated

Fourier-transform infrared spectroscopy (FT-IR, model: PerkinElmer) was used to examine the functional groups expanded on the prepared solids’ surface. Sample disks were scanned over the wavenumber range between 400 and 4000 cm−1 at a resolution of 4 cm−1.

Antimicrobial Effect of Allspice Berry Extract and in Nanoemulsion Form Against Tested Microorganisms

The antimicrobial effect assay of the allspice berry extract in normal and nanoemulsion forms was performed using the agar diffusion method recommended by El-Sayed and El-Sayed (2021) using 1 and 3 µl/ml concentrations. A Mueller–Hinton agar was poured into sterilized plates and allowed to be solidified, and after that, the plates were inoculated using a tested strain concentration equivalent to the MacFarland 0.5 standard. Created wells in the agar layer with a diameter of 0.5 mm and filled with 100 µl of each extract concentration. The plates were incubated at 37 °C/24 h for bacteria and 25 °C/48 h for fungi to subsequently measure the inhibition zone in millimeters. The assays for the extract in normal and nanoemulsion form with each strain were carried out in triplicate.

Ras Cheese Manufacture

Ras cheese was manufactured as described by Hammam et al. (2018) as illustrated in Fig. 2. A blend of cow and buffalo milk was heat-treated at 72 °C for 15 s and quickly cooled to 32 °C. The milk mixture was mixed with calcium chloride (0.02%, w/v) and starter culture (1%) before being left until the titratable acidity was around 0.19%. Milk was renneted, and after complete coagulation for 40 min, the curd was sliced with 0.5-in. knives into vertical and horizontal cubes. The curd was then stirred and gradually heated to 45 °C for 30 min, where it was maintained until the whey acidity reached 0.14%. After draining off about a third of the whey and adding salt (2% milk by volume), the curd was churned again for 5 min. The curd was separated into four equal portions when the whey had completely been drained. The first portion was served as control cheese without nanoemulsion, while other portions as treatments (T1, T2, and T3) were the cheese loaded via allspice berry extract nanoemulsion with different concentrations of (1%, 2%, and 3%), respectively. For 24 h, all curd sections were pressed and molded. The produced cheese was rubbed with dry salt for a week, and it was flipped over every day. After that, cheeses were kept for 4 months at 12 °C ± 2 °C and 80–85% relative humidity. The resultant Ras cheese samples were taken for analysis when fresh, 30, 60, 90, and 120 days of ripening. Three replicates were carried out for each treatment.

Fig. 2
figure 2

Flow sheet diagram for Ras cheese supplemented with microcapsules loaded by allspice berry extract nanoemulsion manufacture

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Physicochemical Properties

The moisture, titratable acidity (TA), and ash contents of the Ras cheese were determined according to AOAC (2012). The pH values were measured using a digital laboratory pH meter (Jenway 3510, UK). The total nitrogen (TN) and soluble nitrogen (SN) of cheese samples were determined by Ling (1963), and total volatile fatty acid (TVFA) was determined in cheese samples according to the method described by Kosikowski (1982); the value was expressed as milliliters of 0.1 N NaOH/100 g cheese.

Determination of Total Phenolic Content (TPC)

The TPC of Ras cheese was determined calorimetrically using a Folin-Ciocalteu reagent (Sigma-Aldrich, Germany) as revealed by Lafka et al. (2007). TPC was calculated using the gallic acid calibration curve (GAE, Sigma-Aldrich, Germany) and expressed as milligrams of GAE per gram.

Determination of Antioxidant Activity

The antioxidant activity of Ras cheese was evaluated as illustrated by Matthäus (2002). Ethanol solutions of Ras cheese extracts (0.1 ml) and 3.9 ml ethanol solution of DPPH (0.0025 g/100 ml CH3OH) were added in a cuvette, and the absorbance at 515 nm (till stabilization) was measured against methanol using a double-beam ultraviolet–visible spectrophotometer Hitachi U-3210 (Hitachi, Ltd., Tokyo, Japan). Simultaneously, the absorbance at 515 nm of the blank sample (0.1 ml methanol + 3.9 ml ethanol solution of DPPH) was measured against methanol. The radical scavenging activity of the Ras cheese samples was uttered as inhibition (%) with the following equation:

$$\mathrm{Inhibition},\%=((\mathrm A^{\mathrm{control}}-\mathrm A_0^{\mathrm{sample}})/\mathrm A^{\mathrm{control}})\times100$$

where A is the absorbance at 515 nm of the control sample and A0 is the absorbance of the tested sample at 515 nm.

Microbiological Evaluation of Ras Cheese Samples Fortified with Microcapsules During the Storage Periods

Ten grams of cheese were grounded and mixed with 90 ml sterilized sodium citrate solution (2%, w/v) under sterile conditions and followed by serial dilution by saline solution (0.9%, w/v) for the microbiological analysis using the pour plate technique. The lactococci counts were enumerated by M17 agar (Oxide) incubated at 37 °C/48 h. For lactobacilli counts, MRS agar (De Man, Rogosa and Sharpe medium, Oxide) was used and incubated at 37 °C/48 h anaerobically. The total bacterial counts were determined by plate count agar with incubation at 35 °C/48 h. Additionally, mold and yeast counts were detected by chloramphenicol rose bengal with incubation at 25 °C/4 days. The spore-forming bacterial counts were detected using plate count agar medium after heating the first dilution with samples for 80 °C/10 min and cooled rapidly with incubation at 35 °C/48 h. The proteolysis bacterial counts were evaluated by plate count agar supplemented with 10% sterile skim milk with incubation at 32 °C/48 h. The coliform counts were detected by violet red bile salt agar with incubation at 35 °C/24 h. All microbiological analyses were done following the standard method of the American Public Health Association (APHA, 1992). All microbiological evaluation counts were done in a triple trial, and the microbiological population was counted as log CFU per gram values.

Sensory Analysis

Sensory analysis was according to Davis (1965). For sensory analysis, 20 members of the Dairy Science Department, National Research Center, Giza, Egypt, and five consumers as a sample from the Egyptian population (all ages 25–55 years old) were asked to contribute to the sensory session to evaluate all Ras cheese samples. The sensory analyses at 0, 30, 60, 90, and 120 storage days were carried out. The selected assessors worked under the direction of the panel leader to develop a sensory language and describe the sensory properties of the products. Samples were evaluated by these trained panelists in terms of flavor, general appearance, and body and texture. Cheese samples were organoleptically scored for flavor (50 points), body and texture (40 points), and appearance (10 points) according to the scorecard.

Statistical Analysis

Statistical analysis was performed according to the SAS Institute (1990) using the general linear model (GLM) with the main effect of treatments. Duncan’s multiple ranges were used to separate among three replicates at p < 0.05.

Results and Discussion

Phenol Compounds for Allspice Berry Extract

Allspice is the most important spice crop and is used extensively as a spice or in folk medicine. Moreover, allspice extract exhibited good biological properties associated with their phenolic compounds such as anti-carcinogenic activity, antibacterial, antihypertensive, and oxygen scavenges (Zhang et al. 2012). Table 2 presents the phenol components in allspice berry extract that were analyzed and quantified by the HPLC analysis. Thirteen components were identified in allspice berry extract. The major compound detected at the highest quantities in allspice berry extract was gallic acid (15,991 µg/g extract), followed by, naringenin (3122 µg/g extract), catechin (2550 µg/g extract), pyrocatechol (2526 µg/g extract), taxifolin (2391 µg/g extract), ellagic acid (2148 µg/g extract), and chlorogenic acid (1657 µg/g extract). Gallic acid has been established to have an anti-cancer effect on various human tumor cell lines by inhibiting the invasion of human melanoma cells and inducing apoptosis in lymphoma cells (Kratz, et al. 2008; Hsu, et al., 2011; Liu et al., 2011; Lo et al., 2011). Also, there are six phenol compounds smaller than 700 µg/g extract such as coumaric acid, vanillin, rutin, syringic acid, methyl gallate, and cinnamic acid. Therefore, allspice extract may serve as a natural functional food ingredient to prevent the progress of many oxidative-related diseases, not only to enhance the shelf life, aroma, color, and taste of the food matrix.

Table 2 Phenol compounds for allspice berry ethanol extract (µg/g extract) by the HPLC profile
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The Morphology of the Prepared Allspice Berry Extract Nanoemulsion

The preparation of allspice berry extract nanoemulsion was carried out using organic allspice extract as well as Tween 80. The structure and morphology of the allspice extract nanoemulsion droplets were characterized via transmission electron microscopy (TEM). Figure 3a displays the TEM image of the allspice extract nanoemulsion formulation representative of the spherical shape of nanoemulsion droplets. TEM also recognized the nano-metric droplet diameter of formulated nanoemulsion. Figure 3b shows the TEM image of encapsulated allspice extract nanoemulsion using oil-in-water nanoemulsion through organic allspice extract (10%) and nonionic surfactant (Tween 80), and the TEM revealed core–shell structure where the allspice extract nanoemulsion has a core with the size around 320 nm, a shell with around 55 nm, and the whole size of the prepared encapsulated allspice extract nanoemulsion at 380 nm.

Fig. 3
figure 3

a TEM image of allspice extract nanoemulsion. b TEM image of encapsulated allspice extract nanoemulsion. c FT-IR of the prepared encapsulated allspice berry extract nanoemulsion

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FT-IR Analyses of Encapsulated Allspice Berry Extract Nanoemulsion (EAEN) Whey Proteins

FT-IR spectrum of encapsulated allspice extract nanoemulsion (EAEN) was recorded in the range from 400 to 4000 cm−1. The identification of (EAEN) is revealed in Fig. 3c, and the comparative location values of specific peaks were designated. The peak appears at 3353 cm−1 related to stretching vibrations of –OH linked to –NH2 in whey protein. Also, the peaks at 2961 cm−1 in addition to 2925 cm−1 are associated with –CH2 groups. The peak at 1658 cm−1 is a representative peak of the main amide group of proteins (–CO–NH2). Moreover, the peak at 1550 cm−1 is the subordinate amide group of proteins (–CO–NH). The peak at 1463 cm−1 and further peaks at roughly 1055 cm−1 relate to –C–O, –C–C, and –C–OH groups. Moreover, the peaks at 1658 cm−1 and 1550 cm−1 are those that are worthy of describing the whey proteins.

Correspondingly, Fig. 3c displays the FT-IR bands of the intrinsic guar gum (GG). The wide bands at 3353 cm−1 matching the stretching vibration of –OH as well as H2O elaborate in hydrogen bonding. A sharp peak that appears at 2925 cm−1 is credited to the –CH stretching vibration. Furthermore, the bands detected in the spectrum between 800 and 1200 cm−1 signified the extremely joined C–C–O and C–OH, besides the C–O–C stretching approaches of the polymer backbone. The peaks about 1658 cm−1 may be ascribed to the ring stretching as well as the bending of H2O molecules because of the hydrophilic nature of GG and also the band at 1658 cm−1 owing to ring stretching of galactose and mannose. The bands about 1463 cm−1 are owing to CH2 bending. Moreover, the peak at 1153 cm−1 is accredited to the glycosidic linkage C–O–C of GG. At 1022 cm−1, a band consistent with CH2 winding was also detected. Also, the FT-IR spectra of dextrin are shown in Fig. 3c, the band at 952 cm−1 and the band look at 721 cm−1 were related to dextrin in the encapsulated allspice extract nanoemulsion (EAEN), and these results demonstrated the dextrin present in the encapsulated allspice extract nanoemulsion.

Antimicrobial Effect of Allspice Berry Extract and in Nanoemulsion Form Against Tested Microorganisms

The antimicrobial effect of the obtained extract in normal and nanoemulsion forms was determined by the well-diffusion agar method at 1 and 3 µ/ml concentrations (Table 3). Antimicrobial testing was performed against seven types of bacteria and two fungi, which are potential intestinal pathogenic microorganisms. The allspice extract in both forms has shown an inhibitory effect on all microorganisms. Among the tested samples, the nanoemulsion form exhibited an inhibitory effect significantly higher than the normal form for all tested strains at two concentrations. The degree of inhibition was diverse depending on the microbial strain.

Table 3 Antimicrobial effect of allspice berry extract normal and in nanoemulsion form
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Generally, the diameter of the inhibition zone ranged between 10 and 17 mm at 1 µ/ml concentration for normal extract and increased to reach between 13 and 19 mm at the same concentration for the nanoemulsion form of extract. The diameter of inhibition was increased at the concentration of 3 µ/ml, which was recorded between 18 and 24 mm for extract in normal form, and the diameter of inhibition was recorded between 19 and 29 mm for extract in nanoemulsion at the same concentration. Commonly, allspice extract is rich in glycosides and polyphenols that display antimicrobial properties (Milenković et al., 2020; Onwasigwe et al., 2017). The activity of extract especially in nanoemulsion form against these strains could be related to the interaction between polyphenol compounds detected in the extract and microbial cell wall. This interaction created pores in the cell wall of strains that lead to the leakage of the cell cytoplasm and cell death (Boyd & Benkeblia, 2013; El-Sayed & El-Sayed, 2021; Lorenzo-Leal et al., 2019).

In addition, the allspice berry extract nanoemulsion in microcapsule form was produced to maintain the stability and the antimicrobial properties when applied in the Ras cheese during the storage period compared with the control cheese. From this point, the microencapsulation efficiency (ME %) for the extract was measured as phenols (the major compounds in the extract) to ensure that the extract nanoemulsion was entrapped inside the resulting capsules. The percentage of ME of the entrapped extract was recorded at 80.97%, which was calculated from the total phenols inside the capsules and the surface phenols on the capsules. This means the used wall materials that involved whey protein, maltodextrin, and guar gum were effective in preserving the extract nanoemulsion as confirmed by Farrag et al. (2018).

Physicochemical Evaluations of Ras Cheese During Ripening

Data in Table 4 expose the physicochemical changes of Ras cheese supplemented by different concentrations of microcapsules loaded with allspice berry extract nanoemulsion during the ripening period. The moisture contents of fresh cheeses range from 39.05 to 41.05%, while, after 120 days, they ranged from 33.51 and 36.51% for cheese treatments. The moisture loss during cheese ripening is mostly due to evaporation. The results showed that the T1 treatment was lower in moisture content than the control treatments, followed by T2 and T3, respectively, during the ripening period. Moreover, there is a considerable difference in moisture content between T3 and other treatments; this may be due to the high ratio of microcapsules in this treatment and the composition of wall materials in the microcapsules.

Table 4 Chemical composition of Ras cheese made by different concentrations of microcapsules during the ripening period
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Moreover, Table 4 demonstrates that the acidity values of Ras cheese treatments were slightly higher than control treatments with opposite trends in pH values. The acidity of Ras cheese which contains microcapsules was significantly (p < 0.05) higher than control cheese during ripening and until the end of the ripening period. Similar results were by Ivanova et al (2021) that showed a slight decrease in the active acidity during the ripening process in kashkaval cheese. Total nitrogen in Ras cheeses supplemented by different concentrations of microcapsules and control cheese is recorded in Table 4. The TN content in all of the experimented Ras cheeses gradually increased with the advancement of the ripening period due to the decrease in the moisture contents in the cheese. Total nitrogen content ranged from 3.19 to 4.59% after 120 days of ripening period in all Ras cheese treatments.

It was noted that there is a significant difference between the treatments at the end of the ripening period. These results were similar to El-Kholy (2015), Hattem and Hassabo (2015), and Abbas et al. (2017) which reported that the TN content was increased pronouncedly (p < 0.05) during the ripening period. This may be due to the corresponding decrease in moisture content and allspice fruit extract and also the presence of proteins in allspice fruit extract (Onwasigwe et al., 2017). The obtained data also indicate that there was a slight increase in ash during the storage period and among supplemented cheese treatments.

Ras Cheese Ripening Indices

Figure 4A shows the soluble nitrogen in the experimental Ras cheese during the ripening period. It is clear from these results in all cheese treatments that the rate of accumulation of SN increased significantly (p < 0.05) with the prolongation of the ripening period. This was attributed to the rate of proteolysis throughout the ripening period. In cheeses supplemented with microcapsules, the SN contents were higher than in control cheese treatment when fresh and until the end of the ripening period. This increase in SN could be due to the proteinases and peptidases released from the allspice berry extract nanoemulsion or soluble proteins from the allspice berry extract nanoemulsion. From the aforementioned results, it could be seen that the addition of microcapsules loaded by allspice berry extract nanoemulsion with traditional starter culture in Ras cheeses led to the increase in SN content, which resulted in higher proteolysis in cheese. These results are in agreement with Tohamy et al. (2011) and Maamoun et al. (2019).

Fig. 4
figure 4

A, B Changes of SN and TVFA contents of Ras cheese fortified by microcapsules loaded with allspice berry extract nanoemulsion during the ripening period. Data expressed as mean of three replicates ± SE. Means with the same capital letters (effect between treatments) are not significantly different. Means with the same small letters (effect of storage) are not significantly different (p ˃ 0.05). Control traditional Ras cheese made without microcapsules; T1, T2, and T3, experiments supplemented with 1, 2, and 3% microcapsules loaded with allspice berry extract nanoemulsion, respectively

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As shown in the results of TVFA, it could be noticed from Fig. 4B the Ras cheese treatments which contain microcapsules were significantly (p < 0.05) higher than the control treatment during the ripening period. The T3 was high in TVFA content followed by T2 then T1 and control treatment which might be attributed mainly to compounds of allspice berry extract and the progress of lipolysis during the cheese ripening. This finding was similar to El-Sayed et al. (2020) who found that the final concentration of TVFA in Ras cheese fortified with jalapeno red pepper was higher than that of control cheese.

Total Phenolic Content (TPC) and Antioxidant Activity of Ras Cheese

TPC and antioxidant activity of Ras cheese fortified with microcapsules loaded by allspice berry extract nanoemulsion during the ripening period are shown in Table 5. Data reveals that control cheese had the lowest content of TPC (6.26 mg/100 g) and antioxidant activity (9.89%) than other cheese treatments. The TPC and antioxidant activity of Ras cheese increased significantly (p < 0.05), and by increasing the percentage of microcapsules, it reached 6.95, 8.80, and 10.00 mg/100 g for TPC and 20.52, 38.28, and 43.64%, respectively, for antioxidant activity, whereas TPC and antioxidant activity were in control, and all Ras cheese treatments decreased during 120 days of ripening period. The presence of phenolic components and other chemical compounds makes allspice a very powerful antioxidant, as numerous of those components are perfect for inhibiting or delaying the oxidation of other molecules by eliminating free radicals from the body. Oxygen-free radicals cause biological damage that can cause healthy cells to mutate, which leads to serious diseases (Zhang et al. 2012).

Table 5 Changes of TPC and antioxidant activity of Ras cheese fortified by microcapsules loaded with allspice berry extract nanoemulsion during the ripening period
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Microbiological Evaluation of Ras Cheese Containing Microencapsulation Allspice Berry Extract Nanoemulsion

All cheese samples and controls were free from the coliform count for the first time and during the storage period of 4 months. This was related to hygienic properties and roles that followed during the manufacturing of cheese. Also, the total lactic acid bacteria that are responsible for cheese flavor and ripening were evaluated as the total counts of lactobacilli and lactococci.

The count of lactococci in all samples gradually declined during the storage period as shown in (Fig. 5), whereas the counts in the zero time were recognized in log 6 cycles with no significance for all samples. After that time, the count significantly declined, and the most dropped was observed in the T2 and T3. The viable lactococci counts were reached at 4.88, 4.41, 3.93, and 3.78 log CFU/g at the end of storage for C, T1, T2, and T3, respectively. The variations between samples during storage would depend on the addition of different concentrations of microencapsulation allspice berry extract nanoemulsion.

Fig. 5
figure 5

Microbiological evaluation of Ras cheese contains microcapsules of allspice extract nanoemulsion during ripening period. Data expressed as mean of three replicates ± SE. Means with the same capital letters (effect between treatments) are not significantly different. Means with the same small letters (effect of storage) are not significantly different (p ˃0.05). Control traditional Ras cheese made without microcapsules; T1, T2, and T3, experiments supplemented with 1, 2, and 3% microcapsules loaded with allspice berry extract nanoemulsion, respectively

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The same trend of results was found in the counts of lactobacilli during the storage period (Fig. 5). In the first zero time, the lactobacilli counts were detected in log 6 cycles without significant differences between treatments and control. At 1 month, the counts were increased slightly, especially in the control sample (reached 7.17 log CFU/g). After that, the counts were dropped gradually to a range between 5.07 and 4.02 log CFU/g at the end of storage (120 days), and a significantly higher decline was observed for T2 and T3.

Moreover, the total bacterial counts were determined in all samples during storage (Fig. 5). The results revealed that the total microbial counts for all samples were significantly lower than control at storage time. At zero time, the total bacterial counts were recorded at log 4 cycles for all treatments, and at 30 days, the counts were increased slightly, but the increase was recorded for control (5.5l log CFU/g). Also, differences in T1, T2, and T3 that were fortified with microcapsules of allspice berry extract nanoemulsion were determined not significant. The total bacterial counts in samples significantly decreased during the storage period than the control, where the counts were recorded at 3.51, 2.89, and 2.42 log CFU/g for T1, T2, and T3, respectively, at the end of the storage period.

Additionally, the mold and yeast counts were not detected for the first time as revealed in Table 6. In the first month of storage, the small mold and yeast counts appeared for control (1.78 log CFU/g) and T1 (0.53 log CFU/g). The mold and yeast counts gradually increased especially for control, where the viable mold and yeast counts were recorded at 4.51, 1.88, 1.48, and 1.11 log CFU/g for control, T1, T2, and T3, respectively at the end of the storage period. Our data revealed that the added allspice berry extract nanoemulsion in microcapsule form enhanced the microbiological safety of cheese during storage. Also, allspice berry extract nanoemulsion was found in microencapsulation form to maintain its stability in the product during storage as mentioned by Mahmoud et al. (2020) and Fayed et al. (2018, 2019). Also, microencapsulation technology permitted the extract to be released in a small amount inside the cheese samples during the storage period which helps to delay the mold and yeast growth, which is related to the antimicrobial effect of allspice berry extract nanoemulsion.

Table 6 Microbiological properties of Ras cheese containing microencapsulation allspice berry extract nanoemulsion
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Also, the proteolytic bacterial counts that were responsible for the cheese ripening were distinguished during storage (Table 6). At zero time, the counts of proteolytic bacterial counts ranged between 1.71 and 2.00 log CFU/g for all samples. During the storage period, these bacterial counts were increased for all samples and were still not significant, but slightly more proteolytic bacterial counts were observed for the control. The proteolytic bacterial counts ranged between 2.73 and 3.13 log CFU/g at the end of the storage period.

Furthermore, the counts of spore-forming bacteria were noted during storage as presented in Table 6. At the first time, at the beginning of storage, spore-forming bacterial counts were not recognized in all treatments. Small amounts of bacterial spore-forming were found for T1 and control at 60 days of storage (1.14 and 1.03 log CFU/g, respectively), while T2 and T3 reported spore-forming counts at 90 days of storage (1.21 and 1.06 log CFU/g, respectively). Also, during the storage period, the count of spore-forming bacteria significantly (p < 0.05) increased, and the highest counts were recorded for control at the end of the storage period. These results might be related to the antimicrobial properties of allspice extract nanoemulsions that were found in microencapsulation form in the cheese samples. Overall, the prepared allspice extract nanoemulsion included in microencapsulation has the ability to preserve Ras cheese’s quality. So allspice extract nanoemulsion in microencapsulated form was recommended to apply for Ras cheese and similar cheeses.

Sensory Evaluation of Ras Cheese Supplemented with Microcapsules During the Ripening Period

The Ras cheese that had 1% microcapsules T1 at zero time and during the ripening period had the highest appearance value, as shown in Fig. 6a. Treatments T2, T3, and control cheese came in second and third, respectively. The rate of acceptance of appearance increased significantly (p < 0.05) as the ripening period was extended. Figure 6b indicates that the body and texture of cheese treatments were preferred at zero time. However, after 120 days of the ripening period, a differential preference in texture was observed among the samples where the T1 cheese sample with 1% microcapsules took the highest value followed by T2, T3, and control samples, respectively. Regarding flavor evaluation, Fig. 6c shows that at zero time, the flavor acceptability of all treatments, T1, was the most acceptable compared with T2, T3, and control, respectively. On the other hand, after 120 days of storage, the flavor preference increased significantly (p < 0.05) for all samples; however, it was still the T1 treatment “highly preferred” for the sample compared to other treatments T2, T3, and control, respectively. From the results, it is clear that the cheese treatments during ripening and at the end of the ripening period with microcapsules possessed the highest value of flavor, while cheese without microcapsules had the lowest. Similar results were obtained by Ivanova et al. (2021), Abbas et al. (2017), and El-Fadaly et al. (2015).

Fig. 6
figure 6

Sensory properties of Ras cheese fortified by microcapsules loaded with allspice berry extract nanoemulsion during ripening period. A Appearance; B body and texture; C flavor; D collective total scores. Control traditional Ras cheese made without microcapsules; T1, T2, and T3, experiments supplemented with 1, 2, and 3% microcapsules loaded with allspice berry extract nanoemulsion, respectively. Data expressed as mean of three replicates ± SE. Means with the same capital letters (effect between treatments) are not significantly different (p ˃0.05). Means with the same small letters (effect of storage) are not significantly different

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Results in Fig. 6d show the general acceptability of cheeses, as total scores were 93.62, 89.06, 86.78, and 86.06 points, respectively, for T1, T2, T3, and control at 120 days of storage. In all cases, the acceptability increased significantly (p < 0.05) during the early stage of the ripening and by extending the storage period. The improvements were slow in cheese without microcapsules, while it was fast in the treatment supplemented with microcapsules. Ripening cheese samples with different microcapsules especially 1% supplement gained the maximum score at the end of a storage period. It could be concluded that the addition of microcapsules of allspice berries extract nanoemulsion can be recommended for the Ras cheese or hard cheese generally.

Conclusion

The extract from allspice berries was typically converted into nanoemulsion drops in nanoform. Additionally, the produced extract was thought to have antibacterial action and be an excellent source of phenol components. The nanoemulsion was obtained in a spherical shape. The impact of allspice berry extract nanoemulsion microcapsules on the microbiological, physicochemical, and sensory characteristics of Ras cheese during a 4-month ripening period determined the microcapsule concentration. As the ripening time progressed, the chemical changes in the Ras cheese supplemented with allspice berry extract nanoemulsion freeze-dried, such as titratable acidity, TN, SN, TVFA, and Ash ratio, generally rose. When compared to other supplemented treatments, the counts of mold, yeast, and spore-forming bacteria in control cheese were the greatest, while the counts of total lactic acid bacteria steadily decreased in all of the cheese samples. Compared to other treatments, the 1% supplementation of allspice berry extract nanoemulsion microcapsules had the highest acceptability score for the sensory parameter. As a result, the Ras cheese business can use the freeze-dried allspice berry extract nanoemulsion to extend the shelf life and improve the functionality of cheese products.