4.2.1. Natural Antimicrobials in Cheese Preparation to Extend the Shelf Life
Table 2 presents the inhibition effects and shelf-life data overviewed relative to spices and plants in different product forms (i.e., polysaccharide compounds, plant extract, essential oils, and liposomes) used as natural antimicrobial alternatives to traditional additives in terms of preserving cheeses obtained by in vitro and in situ assays. What is interesting about the data in this table is that almost all natural antimicrobials reduced the
Staphylococcus aureus count on the surface of Iranian white brined cheese and was at least 6.56% higher than the control cheese with an excellent shelf-life (up to 75 days of storage), as in the case of 15 µL/100 mL of Cumin EO (
Cuminum cyminum), incorporated with the probiotic
Lactobacillus acidophilus at 0.5%
w/
v during cheese manufacturing [
65]. However, despite the outstanding results of 30 µL/100 mL of Cumin EO (CR: 51.36% higher than control), the doses recommended were 15 µL/100 mL once the sensory analysis and organoleptic evaluation were determined to be superior and did not show significant differences in pH values between them [
65]. Afterward, the reduction in
L. monocytogenes counts on Iranian white cheese by Pennyroyal EO (
Mentha pulegium) at a dosage of 0.015% was 24% higher than the control cheese at the end of 60 days of storage [
66]. Despite the better result of antimicrobial effect promoted by the dosage of 0.03% EO (CR of 49% higher), the cheese with 0.015% EO presented more extensive overall acceptability in terms of sensory quality [
66]. The shelf life results can be associated with the high contents of monoterpenoids in these oils, as the major compounds identified by Gas chromatography-mass spectrometry (GC-MS) in Cumin EO were cuminaldehyde (29.02%), α-terpinene-7-al (20.70%), and gamma-terpinene (12.94%) [
65]. By comparison, the major compounds identified in Pennyroyal EO were pulegone (36.68%), piperitenone (16.88%), and 1,8 cineole (14.58%) [
66].
Likewise, the ginger EO (
Zingiber officinale), also rich in monoterpenes and sesquiterpenes, was mainly composed of α-zingiberene (16.1%) and geranial (14.4%), followed by Z-citral (9.2%), β-cedrene (8.6%), geranial acetate (8.4%), α-pinene (6.1%), α-curcumene (5.3%), and α-farnesene (4.4%) [
72]. Thus, preliminary results with 12% (
v/
v) ginger EO demonstrated an in vitro bactericidal effect against pathogenic strains (
Pseudomonas aeruginosa,
Salmonella enterica ser. Typhimurium,
S. aureus,
E. coli O157:H7,
L. monocytogenes, and
S. aureus) once the MBC/MIC was two with an inhibition zone ranging from 13–37 mm [
72]. The quality parameters of cheese were not evaluated.
The ethanolic extract of dry cedar leaves (
M. oleifera) was rich in nutritional and phenolic components and was suggested as supplemental nutrition and preservative agent to extend the shelf life of cream cheese. The amount of 4.00 mg of cedar ethanolic extract (95%) showed significant in vitro antimicrobial action against pathogenic strains (e.g.,
Bacillus cereus,
Bacillus subtilis,
S. aureus,
P. aeruginosa,
L. monocytogenes,
Escherichia coli,
and Salmonella) with inhibition zone diameter ranging from 15–22 mm. At the same, the concentration per milliliter of milk in the preparation of cream cheese, the coliforms, yeasts, and molds growth inhibition in cream cheese was 100% once any count was observed during the 4 weeks of storage, contrary to the negative control sample [
42]. For quality, besides the addition of
M. oleifera extract improved sensory parameters of taste and flavor, the color changed due to dietary fibers’ presence.
The fennel (
Foeniculum vulgare Mill) is a distribution plant in Central Europe and the Mediterranean with antimicrobial and antioxidant properties [
67,
76]. Due to these properties, Caleja et al. (2015) [
67] reported a useful in vitro antimicrobial activity of fennel phenolic-enriched extract against
B. cereus,
S. enterica ser. Typhimurium,
Aspergillus niger, and
Aspergillus versicolor by MIC values ranging from 0.02 to 0.75 mg/mL, with the
S. enterica ser. Typhimurium and
B. cereus are the most sensitive to fennel extract by the MIC values of 0.035 and 0.02. Furthermore, cottage cheese preparation with fennel extract improved antioxidant properties 62.96% greater than the control cheese and up to 14 days of storage without degradation signals [
67].
Black cumin (
Bunium persicum) is a typical plant in Afghanistan and Iran’s dry temperate regions [
77] that was chemically characterized and presented cuminaldehyde (11.4%) as the most abundant bioactive compound in its oily seeds [
68], which are frequently associated with good antioxidant and antibacterial properties. Thus, Ehsani et al. (2016) [
68] evaluated EO’s antimicrobial capacity from the seeds of black cumin against
L. monocytogenes and
E. coli O157: H7 on Iranian White Cheese. At the end of the 45 day storage, the
L. monocytogenes and
E. coli count reduction was 7–9% and 6–9% higher than the control, respectively, by low EO doses from 1–2% (
w/
v) [
68]. Furthermore, the black cumin EO showed higher sensory quality attribute scores (e.g., color, texture, flavor, odor, and general acceptability) than the control.
The pink pepper EO (
Schinus terebinthifolius) is widely distributed in South America (Brazil, Paraguay, and Argentina) and has been reported with high in vitro and in situ antimicrobial activity in several food matrices. An inhibition zone ranging from 39 to 97 mm was reported by in vitro antibacterial assay against
B. cereus at a MIC of 0.85 mg/mL-EO from mature fruits, and it was better than green fruits. Its biopreservative potential in situ by low doses (0.7–2% of EO-mature pulp) was also demonstrated for fresh Minas-type cheese against
L. monocytogenes extending the shelf life up to 30 days of storage, with a count reduction 11–18% higher than the control cheese [
70]. Despite this, the authors mentioned its potentially undesirable effects on sensory quality.
As discussed before, basil oil (
O. basilicum L.) is another essential oil rich in terpenes and phenolics (e.g., linalool and estragole) that stands out as a preservative due to its antimicrobial and antioxidant properties [
43,
44]. Likewise, LAB can also produce substances (e.g., enzymes, bioactive lipids and peptides, vitamins, and exopolysaccharides) that inhibit pathogenic microorganisms and improve dairy products’ protection and functional properties, in addition to its role in milk fermentation [
78]. Thus, its characteristics might explain the synergistic effect of 5% basil oil and different LAB strains as antimicrobial in unripened goat cheese during 120 h of storage by 100% inhibition of
Enterobacteriaceae growth compared to the control cheese [
44]. Moreover, there was no significant change in the product’s pH; the texture with the addition of basil and LAB (
L. brevis) was 1.3% lower when compared to the control cheese; as for colors, the L* values of cheese containing basil-LAB was lower, while a * and b * was higher than the control cheese; and for sensory quality, cheese containing basil-LAB had higher acceptability (5 points on the hedonic scale) than the control cheese (4 points) [
44].
The identification of carvacrol (80.9%) and ρ-cymene (7.7%) as the significant constituents of thyme EO (
Thymus algeriensis) seems to be a primary determining factor of its antioxidant and tremendous broad-spectrum in vitro antibacterial and antifungal activity reported by Bukvicky and colleagues (2018). These authors related expressive values of MIC ranging from 0.03 to 0.09 mg/mL, and MBC ranged from 0.05 to 0.15 mg/mL against Gram negative and Gram positive bacteria (
S. aureus,
S. enterica ser. Typhimurium,
E. coli,
P. aeruginosa,
L. monocytogenes,
Micrococcus flavus, and
B. cereus), as well as the impressive MIC of 0.01 mg/mL against
Aspergillus spp.,
Trichoderma spp., and
Penicillium spp. [
71]. In terms of cheese shelf life, this study found a reduction in
Penicillium aurantiogriseum contamination incidence (65% higher than control sample) on sliced soft cheese after 30 days of storage at 4 °C by the use of thyme EO at a concentration of 25 µL [
71]. Concerning sensory quality, the 15 µL EO improved color and texture parameters but did not stands out in terms of flavor compared to the control cheese [
71].
The intense preservative action of ε-poly-lysine against
L. monocytogenes on the Grana Padano cheddar cheese surface at a low dosage (from 0.05 to 0.2 mg/mL) was 30–100% higher than control cheese after 15 days of storage at 4 °C and 25 °C [
64].
In addition to ginger (
Z. officinalis), garlic (
Allium sativum L.) has also stood out in terms of several biological properties such as antimicrobials relative to preserving cheese, although the sensory quality can be affected for both level and type of spice. Recently, Salih et al. (2019) [
73] evaluated the antimicrobial effect of garlic and ginger powders at concentrations ranging from 2% to 6% on white cheese storage for 14 days at 4 °C. The authors reported that any count of Coliforms and
Salmonella was detected for the lowest content (2%) of booth garlic and ginger powder during the period of storage. By contrast, garlic powder showed a more potent action than ginger powder against fungi and yeasts since, when applied at 2–6% concentration, its count reduction was 58.6–79.3% higher than the control cheese [
73]. Regarding the sensory quality, in addition to the 2% ginger-cheese having better results in terms of overall acceptability than the control cheese, all samples with garlic powder had greater overall acceptability (taste, flavor, and texture) by the consumer (as well as pH) than the control or ginger powder [
73]. However, all treatments showed lower color scores than the control [
73].
Recently, the addition of combining terpene-rich oregano EO (0.03 µL/mL) and rosemary oil (1.32 µL/mL) during cheesemaking was suggested as a natural preservative to fresh cheese during refrigerated storage once the synergistic effect between them for inhibiting
E. coli O157: H7 growth was 68% higher than the control cheese during 21 days of storage [
45]. Furthermore, the quality parameters were also improved once the cheese containing both EO showed lower hardness and better results of softness and chewiness and similar values for L * and b * in color compared to the control cheese. Until the end of 21 days of storage, the authors detected eucalyptol, camphor, and α-pinene, which produced a refreshing aroma, minty flavor, and softness to fresh cheese in addition to explaining the shelf life results obtained [
45].
The application of the gaseous phase of Lemon leaf EO (
Citrus limon var
Pompia) in ricotta Salata cheese slices showed a tremendous antimicrobial activity against
L. monocytogenes by a MIC of 0.086 µL/cm
3, which is probably related to phytochemicals screened as linalyl acetate, limonene, and two Citral isomers—neral and geranial [
75]. Thus, low doses of gaseous phase-lemon EO, ranging from 0.05 to 1 mL, resulted in a decrease in the
L. monocytogenes count of 21–60% higher than the control Ricotta Salata cheese after 30 days refrigerated storage (at 5 °C). In addition to that, the natural antimicrobial did not cause a significant difference in the LAB and total mesophilic bacteria (TMC) [
75].
The evaluation of green propolis ethanol extract (EEP) (
Apis mallifera) as a cheese preservative was motivated by its tremendous in vitro bactericide and fungicide effects against several yeasts and bacteria (
Staphylococcus,
Bacillus,
Enterococcus,
Corynebacterium, and
Proteus spp.) by MFC/MIC ranging from 1 to 2.2 and MBC/MIC equal 2, respectively, with the most sensitive being
S. cerevisiae (MFC = 0.63%) and the most resistant fungi being
Candida parapsilosis (MFC = 5%) [
74]. Thus, a dosage from 1.25% (
w/
w) EEP was capable of completely inhibiting
Staphylococcus saprophyticus, and the
Staphylococcus equorum count after 24–28 h, 37 °C, as well as from 1.25% or 2.5% (
w/
w) completely inhibited
Yarrowia lipolytica or
Debaryomyces hansenii after 48–72 h (for yeasts) at 37 °C on the surface of Gorgonzola-type cheese. However, only 5% EPP was suggested as a promising concentration once it showed a sensory quality (i.e., taste, odor, and overall) similar to the control cheese, and 10% EPP was worse than the control [
74].
Despite the potential antimicrobial effect of several essential oils in extending the shelf life of cheeses, certain oils have low solubility, photosensitivity, and high volatility and can negatively interfere in the milk matrix by reducing its sensory quality [
79]. Thus, these disadvantages contribute to studying these oils in the encapsulated form in order to prevent adverse effects on the cheese’s quality or physicochemical properties. Despite lemongrass oil (LO) (
Cymbopogon citratus) decreasing the physical and sensory quality of cheese at the end of 15 days of storage, LO encapsulated in a liposome (1 mL·LO/100 g) promoted a count reduction in
L. monocytogenes on Kerrygold Cheddar 59% that was higher than free LO and the control, without affecting the color surface, texture, and sensory quality (e.g., aroma and taste) of cheese at the end of storage [
69].
4.2.3. Natural AntimicrobialsinActive Coatings/Nanopackaging for Preserving Cheese
Closer inspection of
Table 4 shows natural antimicrobials as active ingredients of coating and packaging with a broad-spectrum microbial inhibition higher than control cheese.
If the ratio MBC/MIC ≤ 4, the effect was considered bactericidal, but the effect was defined as bacteriostatic if MBC/MIC > 4 [
82]. Thus, most of the studies overviewed revealed higher bactericidal and bacteriostatic effects with prolonged shelf life (>60 days of storage) at low doses of the antimicrobial compound. The addition of pink pepper EO as a bioactive ingredient into film packaging at a concentration of 5.45 mg/cm² showed 100% antimicrobial activity against
S. aureus and
L. monocytogenes in sliced mozzarella cheese during the 12 days of storage, once no count was detected [
82]. The acetate cellulose film was chosen for packaging due to its capacity to form films at low temperatures, avoiding EO’s volatilization during polymer film processing [
82]. Moreover, the in situ assay also demonstrated the affinity between EO’s non-polar components with lipids of cheese, allowing EO migration from the film to cheese. The authors suggested that it could benefit the application of active packaging by direct contact [
82]. This strategy is promising for eliminating the disadvantages of undesirable effects on the product’s sensory quality when the direct addition of EO occurs during cheesemaking [
70].
A novel nanopackaging of a bio-nanocomposite film made from 0.92% (
w/
w) chitosan, 0.92% (
w/
w) cellulose gum, and 2–8% (
w/
w) zinc oxide nanoparticles (CS/CMC/ZnO) enhanced the shelf life of Egyptian white soft cheese completely inhibited with coliforms, TBC, yeast, and molds and reduced pathogenic strain contamination (
S. aureus,
Bacillus spp.,
P. aeruginosa,
E. coli, L.
monocytogenes,
C. albicans, and
A. niger) with IZ values ranging from 8 to 15 mm during 30 days of storage at 7 °C [
85]. Regarding cheese quality, contrary to the control cheese, the characteristic color of fresh cheese was preserved, and the moisture, pH, and titrable acidity were preserved without molds during storage period in cheese packaged by bionanocomposite film [
85].
Preliminary results of in vitro antibacterial effect of ginger EO reported MIC of 2.3 µL/mL and MBC of 4.7 µL/mL for
L. monocytogenes and
E. coli O157:H7 as the most sensitive MO, as well as CR: 20–40%
L. monocytogenes by micro-atmosphere (at 0.12 to 0.35 µL/cm
3) due to the action of volatiles from EO [
72]. Ginger EO as a nanoscale antimicrobial encapsulates in the protein ultrafine fibers (12%
v/
v; EO/polymer), purposing active packaging of the Minas-fresh cheese, and reduced
L. monocytogenes contamination after 12 days of storage with the count reduced to 10% higher than the control. The cheese’s physical-chemical and sensory parameters were not evaluated in the study in question [
72].
Pomegranate peel extract (PPE) in zein films has become an alternative to Himalayan cheese (Kalari) packaging. In the study of Mushtaq et al. (2018) [
86], the PPE concentrations of 25, 50, and 75 mg/g of a film-forming solution were evaluated for their in vitro antibacterial activity of IZ until 20 mm (101 to 123% higher than the control) against
E. coli,
P. perfringens,
Micrococcus luteus,
Enterococci faecalis,
S. aureus,
Proteus vulgaris, and
S. enterica ser. Typhi. Regarding shelf life on day 21 of storage by in situ assay using the Kalari as cheese model, the PPE concentration range of 25–75 mg/g showed a reduction in the microbial counts of 54–73% and 71–100% for total bacterial count and yeasts and molds, respectively, which are higher compared to the control cheese [
86]. The sensory analysis performed during the tenth day of storage on cheese containing 75 mg/g PPE was preferred in the evaluated items (i.e., appearance, flavor, aroma, bitterness, and overall acceptability) compared to the control cheese [
86].
Another critical finding was other natural antimicrobials from the protist kingdom such as sodium alginate or alginic acid, a polysaccharide obtained from brown algae, and its association with essential oils with consumer appreciation in sensory quality. Sodium alginate as a coating additive containing 3% (
w/
v) potassium sorbate inhibited the
Pseudomonas spp. and other psychrotrophic microorganisms growth in fresh mozzarella cheese, with an efficacy 12% higher than the control cheese during 8 days of storage (at 8 ± 1 °C), preserving the sensory quality compared to the control that was refused after 4 days [
84]. Recently, 0.2% sodium alginate’s effectiveness as a coating additive for Béja Sicilian cheese preservative was confirmed with the advantages of replacing the chemical additive with 3%
Pimpinella saxifrage EO as the active ingredient [
87]. The most exciting finding was that despite the strategy improving bacterial stability by inhibiting three-gram positive (
B. cereus,
M. luteus, and
L. monocytogenes) and three-gram negative (
E. coli,
P. aeruginosa, and
S. enterica ser. Typhimurium) bacteria by bactericidal and bacteriostatic action, the weight loss, color, pH, and oxidative stability were also preserved without flavor impairment during 60 days of refrigerated storage [
87]. Additionally, Ksouda et al. (2019) [
87] also assessed
Pimpinella saxifrage EO’s acute toxicity, showing that doses of 250 and 500 mg/kg had no harmful effects on the mice model. The presence of the flavoring compound anethole, eugenol isomers, and p-anisaldehyde as main compounds in
P. saxifrage EO might explain its great results beneficial to cheese preservation.
The effect of lactic acid (LA) and chitooligosaccharide (COS) as active ingredients for edible-whey protein isolate film was compared to PVA-commercial coating containing natamycin as an active ingredient applied on the surface of Portuguese cheese during 15 days of storage [
83]. While natamycin showed the highest efficacy against yeasts and molds, the edible coating formulated with LA and COS showed a potency of 98% and 100% count reduction higher than the control against
Pseudomonas and
Staphylococcus species [
83]. Natamycin also has been used as an active ingredient in active bio-packaging to preserve cheese during storage, despite being effective only against fungi. For example, natamycin was an active ingredient of active packaging made from tapioca starch film (at 9.25 mg/dm
2 film) and preserved Port Salut cheese against
S. cerevisiae with an efficacy 62% higher than samples using natamycin applied by spraying technique, during 216 h of storage at 25 °C [
59]. Afterward, recent investigations by Seydim and colleagues (2020) evaluating the effect of oregano 2% EO, garlic EO, nisin, or natamycin as active ingredients of whey protein isolate film (WPI) and natamycin observed the highest reduction count (until 20% higher than control) against
Penicillium spp., while nisin had the highest reduction count (until 25% higher than control) against
L. monocytogenes during 15 days of storage. On the other hand, the films containing oregano and garlic EO stand out against
E. coli O157:H7 strains, with 40% efficacy higher than the control at day 15 of storage [
88]. However, the investigations citing natamycin and nisin as active ingredients of bio-packaging for preserving cheese did not analyze the impact on cheese quality.
4.2.4. Combined Methods: Natural Antimicrobials and Packaging Conditions in Non-Thermal Treatments
Table 5 displays the effect of combined treatment on extending the shelf life of cheese and microbial count reduction percentage. What is striking about this table’s data is that while lysozyme/Na
2-EDTA salt does not affect the inhibition growth of
Pseudomonas species when burrata cheese was packaged in air, the combination of them with modified-atmosphere packaging in the
Pseudomonas spp. Showed that count reduction was at least 22–39% higher than the control during 7 days of storage [
89]. Unfortunately, both the MAP and lysozyme used affected the sensory attributes such as texture and overall acceptability of cheese [
89].
Likewise, the pine needle extract (
Cedrus deodora), rich in acidic and phenolic compounds, was also suggested as a natural preservative for cheese due to improved oxidation stability and storage quality of low-fat Kalari cheese treated with 2.5–5% pine needles extract, dried, and aerobically packaged in low-density polyethylene bags [
90]. From the minimum concentration, the authors have already observed favorable microbiological results during the 28 days of storage at 1 ± 4 °C once coliform growth was not detected in any sample (the milk for cheese manufacturing was heat-treated at 90 °C), while an antifungal effect at least 17% higher than the control cheese was reported for samples treated with extracts against Yeasts and Molds. Furthermore, this research also reported a reduction until 17% and 7% higher than the control cheese in the count of psychrophilic bacteria and total plate count, respectively [
90]. The pine needles extract improved the Kalari cheese physicochemical parameters once both doses of 2.5% or 5% at the pH of 4.35 or 4.18, respectively, were below the control cheese pH (4.71) at the end of the 28 days of storage; [
90]. Furthermore, sensory quality parameters (e.g., appearance, color, flavor, texture, sourness, and overall acceptability) have been preserved [
90].
First, unlike the sodium benzoate in
Table 1, citric acid as an active ingredient of tapioca flour packaging (10 g/100 g) treated with pulsed light against
L. innocua on sliced cheddar cheese (
Table 5) was 36% higher than the control during storage; however, the product quality (e.g., pH, moisture, and mechanical properties) changed after 7 days of refrigerated storage [
26]. Nevertheless, the adjustment of citric acid content is promising once the antioxidant effect prevented cheddar cheese color instability due to lipid oxidation [
26].