АннотацияPlants have been used as food additives worldwide to enhance the sensory qualities of foods and extend their shelf life by reducing or eliminating foodborne pathogens. They also serve as therapeutic agents due to their beneficial effects on human health through their anti-cancerous, anti-inflammatory, antioxidant, and immune-modulatory properties.
Plants can be added to food as a dry powder, grated material, paste, juice, or as an extract that can be produced by a variety of methods. Plant extracts and essential oils are concentrated sources of bioactive phytochemicals that can be added to food in small amounts in a variety of forms. These forms include liquid, semi-solid, or dry powder for easy and uniform diffusion. Encapsulation can protect bioactive compounds from temperature, moisture, oxidation, and light, as well as allow for controlling the release of the encapsulated ingredients. Nanoemulsions can enhance the bioactivity of active components.
This review explains how plant extracts and essential oils are used in the dairy industry as antimicrobial materials, analyzing their impact on starter bacteria; as natural antioxidants to prevent the development of off-flavors and increase shelf life; and as technological auxiliaries, like milk-clotting enzymes, stabilizers, and flavoring agents. Therefore, plant extracts and essential oils are a better choice for the dairy industry than plants or their parts due to a wide range of applications, homogeneous dispersion, and ability to control the concentration of the bioactive ingredients and enhance their efficiency.
Ключевые словаPlant extracts, essential oils, dairy products, natural antimicrobials, milk-clotting enzyme, natural antioxidants
Plants have been utilized as medicine and preservatives, as well as food flavorings, since ancient times. They are used fresh or dry and have many different forms of use, including plant parts such as leaves, roots, flowers, seeds, crusts, tubers, or herbs. They are also applied in crushed or ground forms, or as extracts prepared in different ways . Plants rich in secondary components (alkaloids, organosulfur compounds, glycosides, flavonoids, tannins, phenols, coloring agents, and resins) are classified as medical and aromatic plants . Medical and aromatic plants are used for aromatic, coloring, preservative, and antioxidant purposes as spices and food additives. Phenolics are free radical eliminators and metal chelators. They can inhibit the lipid peroxidation and exhibit various physiological activities as antioxidants. Many foodborne pathogenic and spoilage bacteria, as well as molds and yeasts, can be inhibited by phenols and extracts rich in such substances . In addition, they have potential in the prevention and treatment of some chronic diseases, including cancer, diabetes, and cardiovascular disease . Essential oils (terpenes, esters, alcohols, ketones, aldehydes, and phenols) are of great interest to the food and cosmetic industries, as well as medicine, due to their wide range of biological properties (antimicrobial, antifungal, antioxidant, anti-inflammatory, insecticide, analgesic, anticancer, cytotoxic, etc.) .
Milk and dairy products are the most common foods in the diets of all age groups. Their popularity can be attributed to milk’s unique components and properties, as well as the fact that a wide variety of foods can be prepared using this ingredient. The market for valueadded functional foods has expanded manifold due to the consumers’ increased awareness of, and interest in, following healthy dietary strategies to achieve health benefits from foods beyond their basic nutrition . With the introduction of fortified foods, there has been a surge in using plants and their extracts as valuable additives in dairy products because they contain numerous bioactive components that perform a variety of functions. Additionally, plants have been used for centuries in milk and dairy products as technological auxiliaries (e.g. milk-clotting enzyme preparations, cheese wrappers) or as natural preservatives due to their antimicrobial and/or antioxidant properties .
Plant extracts are concentrates of bioactive phytochemicals obtained through extraction technologies including steam distillation, cold pressing, or solvent extraction, with or without pretreatment. In addition to classic approaches, new extraction methods have also been investigated, such as ultrasonic, microwave, and supercritical fluid extraction. Essential oils can also be extracted from various parts of plants, depending on the species and variety . The purpose of extraction is to obtain the largest quantity of bioactive components from plants. With the help of solvents, soluble plant metabolites are separated from the insoluble cellular marc. After the solvent is removed, the products can be used in liquid, semi-solid, dry-powdered, or encapsulated form. Nowadays, plant extracts are increasingly becoming important additives in the dairy industry due to their high content of bioactive compounds that have antimicrobial and antioxidant activity . The aforementioned compounds also delay the development of off-flavors and improve the shelf life and color stability of food products. Due to their natural origin, they are excellent candidates to replace synthetic compounds, which are generally considered to have toxicological and carcinogenic effects . Additionally, plant extracts can be added to food in low concentrations with efficient distribution and uniformity. Encapsulation protects them from temperature, moisture, oxidation, and light, while allowing scientists to control the release of encapsulated ingredients, as well as to mask off-flavors and unpleasant odors . Therefore, we mainly aimed to review the possible uses of plant extracts as antioxidants, antibacterials, and antifungals in the dairy industry, as well as to study their effects on the starter bacteria. Furthermore, we analyzed the use of plant extracts instead of renin enzyme to coagulate milk and determined their effect on the yield of the resulting cheese, as well as its physiochemical and sensory properties.
РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ
Plant extracts as natural antimicrobials. Mode of action. Antimicrobials are compounds used for food preservation by controlling the growth of spoilagecausing and pathogenic microorganisms. There are a range of natural compounds with antimicrobial activity that have been identified from various sources (plants, animals, or microbes). However, due to the structural differences between Gram-negative and Grampositive bacteria, the efficacy of antimicrobial agents mayvary . In plants, natural compounds exerting antimicrobial activity are phenolic compounds, alkaloids, sulfur-containing compounds, and terpenoids, as well as essential oils and their constituents [12–15]. Generally, natural compounds with different chemical groups can permeate or disrupt the cytoplasmic membrane, allowing the passage or release of nonspecific compounds. Increased cell membrane permeability leads to the release of intracellular compounds, especially potassium, calcium, and sodium ions, causing irreversible damage [14, 16]. Natural compounds may also inhibit the ATPase enzyme responsible for the energy generation of the cell, which leads to cell death [8, 17].
In a study by Gonelimali et al., the plant extracts significantly affected the cell membranes of Grampositive and Gram-negative bacteria, as demonstrated by the decline in pH and the hyperpolarization of the cell membrane . Some fatty acids, in particular, have the ability to interfere with the structure of the cell membrane, displacing phospholipids and increasing their permeability. Five of them are acetylenic: 6-hexadecinoic, 6-heptadecinoic, 6-octadecinoic, 6-nonadecinoic, and 6-eicosinoic acids, whereas the other three are saturated: palmitic, heptadecanoic, and stearic acids . The activity of polyphenols depends on the number and position of hydroxyl groups. Polyphenols may inhibit the enzymes of microorganisms, possibly through interactions with sulfhydryl groups or through less specific interactions with proteins . Miklasińska-Majdanik et al. reported that phenolic compounds partially damaged the bacterial membrane, inhibited virulence factors such as enzymes and toxins, and suppressed bacterial biofilm formation . In addition, some natural polyphenols, aside from their direct antibacterial activity, exert a synergistic effect when combined with common chemotherapeutics.
Essential oils also have antibacterial, antioxidant, and antimutagenic properties, as well as possible health benefits. These natural compounds, which are generally recognized as safe (GRAS), reduce lipid oxidation in foods and hence hold potential as natural food additives . There are many types of compounds in essential oils which have proven antimicrobial properties. They include phenolic monoterpenes (thymol and carvacrol), phenylpropanoids (eugenol), alcoholic monocyclic monoterpenes (α-terpineol and terpinen-4-ol), as well as bicyclic monoterpene hydrocarbons (α-pinene) and ketones (camphor). The acidic nature of the hydroxyl group of phenols facilitates a hydrogen bond with the enzyme active center, which is responsible for their high activity . Terpenoids can rupture the cell membrane due to their lipophilic nature.
Challenges of using plant extracts as antimicrobials in the dairy industry. In vitro, several studies have demonstrated that plant extracts and essential oils of aromatic and medicinal plants have antimicrobial activity against pathogens and spoilage microorganisms associated with food contamination [23, 24]. However, these results cannot be strictly valid due to the complexity of the food matrix. Moreover, many factors interfere with the activity of these compounds, such as proteins, lipids, packaging, storage temperature, type of microorganism, and compound stability [17, 25]. Proteins and lipids, for example, can wrap around the surface of the microorganism, forming a physical barrier that prevents the bioactive compound from coming into contact with the microorganism, thus reducing its efficacy . In a similar report, the antimicrobial activities of cinnamon and clove essential oils were lower in the high-fat milk samples than in the skim milk samples . Dairy products are foods with a high content of proteins, lipids, minerals, and vitamins. So, when adding extracts and essential oils of aromatic plants to dairy products as antimicrobials, we must take into account the following factors:
1. Natural compounds added as antimicrobials should be in greater concentrations than those tested in vitro. According to Gammariello et al., the concentration of active compounds used to inhibit the growth of pathogenic microorganisms in Fior di Latte cheese was significantly higher than the level tested in vitro . The minimum inhibiting concentration of pomegranate essential oil against Listeria monocytogenes and Staphylococcus aureus (105 CFU/mL each) was higher than 2.5 mg/mL in a culture medium, while its concentration of 40 mg/mL in Cheddar cheese failed to inhibit the same population of those microorganisms . Hassanien et al. also mentioned that the 0.1% concentration of black cumin essential oil reduced the growth of L. mononocytogenes, S. aureus, Escherichia coli, and Salmonella enteritidis in a culture medium, while in cheese, such concentrations were not effective against S. aureus and L. monocytogenes ;
2. In some cases, mixing some plant components at low concentrations has a higher antimicrobial effect than adding them separately, which proves their synergistic effect ;
3. Dairy products contain all the nutrients necessary for the microbial growth of cultured cells, allowing for a faster recovery of cells damaged by natural antimicrobials ;
4. The contents of natural compounds can decrease during processing and storage. Libran et al. reported a decrease in the content of compounds from basil and tansy essential oils added during cheese production . In another study, rosemary essential oil added to sheep milk during cheese manufacturing had a loss of 37.49% ; and
5. Microencapsulation can improve the stability of natural substances throughout processing and storage . Nanoemulsions of plant extracts can decrease the quantity of a required effective dose and enhance the material’s bioactivity against bacteria by allowing them to penetrate the cell membrane and thus destabilize its lipid bilayers . For example, a nanoemulsion of anise extract performed better than bulk extract as an antimicrobial agent against some foodborne pathogenic bacteria .
Plant extracts as antibacterials in dairy products. Several plants in various forms (powder, essential oils, extracts, etc.) have been successfully used in dairy foods. Plant extracts of cinnamon, garlic, lemongrass, cress, rosemary, sage, and oregano individually inhibited the population of L. monocytogenes in processed cheeses . According to Shan et al., all the extracts of cinnamon stick, pomegranate peel, grape seed, oregano, and clove inhibited the growth of S. aureus, L. monocytogenes, and S. enterica in cheese . As a result, these extracts, especially clove, have the potential to be employed as natural food preservatives. Cayenne and green pepper extracts also reduced the S. aureus population in Egyptian Kareish cheese . Mahajan et al. reported that the aqueous extracts of pine needles improved the microbiological properties of low-fat Kalari, an Indian hard cheese, due to their antioxidant and antimicrobial properties . Sulfurcontaining compounds are credited with antimicrobial activity in plant-based compounds, particularly diallyl sulphides in Allium species, terpenoids (carvone and limonene) in spearmint essential oil, eugenol in clove oil, and thymol in thyme oil. Ginger’s antimicrobial activity is attributed to several compounds, including gingerols, gingerdiols, and shogaols [37–39]. Table 1 lists bioactive components and their functional qualities in plants used to manufacture functional dairy products.
In addition, the essential oils of aromatic plants also showed anti-bacterial activity in food preservation, even with Gram-negative bacteria. Gram-negative bacteria have an effective permeability barrier consisting of a thin lipopolysaccharide exterior membrane, which could restrict the penetration by the extruding plant extracts. Gram-positive bacteria have a mesh-like peptidoglycan layer which is more accessible to permeation by plant extracts . In Feta cheese and Iranian white cheese, oregano (0.1%) and thyme (0.1%), salvia (0.1%), basil (1%), and black cumin essential oils had antimicrobial activity against L. monocytogenes [41, 42]. In Iranian white cheese inoculated with E. coli O157:H7 and treated with black cumin essential oil, the pathogen growth was significantly lower compared to the control during storage . Adding clove essential oil to Paneer cheese increased its shelf life to 10 days in the treated cheese compared to 5 days in the control sample. Furthermore, the control samples had a higher microbial count compared to the treated cheese. Clove essential oil added at concentrations of 0.5 and 1% dramatically reduced the growth rate of L. monocytogenes in cheese at 30 and 7°C. However, high concentrations of clove oil may adversely affect the sensory properties of food. Thus, small concentrations may be enough to ensure low bacterial load and, therefore, food safety .
The addition of aqueous licorice and cinnamon extracts to yoghurt exhibited the strongest inhibitory effect on Helicobacter pylori development when compared to the control yoghurt . According to Mahgoub et al., adding 0.2% Nigella sativa essential oil to the cheese improved its physicochemical and sensory qualities. In addition, it provided the most effective antibacterial capability against S. aureus, S. enteritidis, and E. coli . In goat milk-based yoghurt containing Lactobacillus acidophilus and roselle extract, higher antimicrobial activities were observed against Bacillus cereus, E. coli, S. aureus, and Salmonella typhi. This could be attributed to the production of higher antimicrobial compounds such as antimicrobial peptides and organic acids .
Plant extracts as antifungals in dairy products. Fungi are spoilage microorganisms that grow in foodstuffs during storage, reducing their nutritional value and sometimes producing mycotoxins. As a result, foods become unfit for consumption . The growth of fungi on the cheese surface can be inhibited by using some plant-based compounds. For example, cinnamon leaf and bark essential oils (≤ 10% (v/v) showed the highest antifungal activity during the ripening of Appenzeller cheese . Also, incorporating cinnamon oil with 5% cinnamaldehyde into a film coating of spreadable cheese delayed the growth of Aspergillus niger and Penicillium expansum . Molds and yeasts were not detected in UF-soft cheese fortified with ginger and garlic extracts until the end of storage, 42 and 90 days, respectively [37, 50]. Sağdıc et al. found that garlic and thyme extracts inhibited most molds and yeasts in soft cheese . Plant extracts can also help to delay or prevent the formation of mycotoxins. Vazquez et al. found that eugenol (200 µL/mL) added to Arza Ulloa cheese reduced the synthesis of citrinin, a toxin generated by Penicillium citrinum . Sindhu et al. also found that the essential oil isolated from curcuma leaves (1.5%) inhibited aflatoxin formation . At concentrations of 0.50–1.5%, the oil of Satureja hortensis L. exhibited antibacterial activity, while its alcoholic extract had no effect on S. aureus mycelia growth. Similarly, the S. hortensis essential oil implanted in fresh cow’s cheese prevented S. aureus growth, but its ethanol extract did not appear to be effective . Labneh, a concentrated yoghurt with 0.2 ppm essential oils of thyme, marjoram, and sage, had a 21-day shelf life compared to the control, with yeast and mold observed in the control from the 14th day onwards . Labneh containing 0.3% cinnamon oil, on the other hand, had a longer shelf life (8 days) when stored at 6°C compared to the control product .
Effect of plant extracts on starter culture activity. Starter cultures are responsible for fermentation and provide the desired sensory qualities to the finished product. Plant extracts and essential oils, which are intended to suppress pathogenic bacteria, prevent spoilage, or improve sensory characteristics, have been demonstrated in numerous studies to have no effect on the activity of starting cultures. Lactic acid bacteria are the most resistant bacteria to antimicrobial agents in plant extracts and essential oils at concentrations that limit the growth of pathogenic microorganisms . In particular, the count of lactic acid bacteria in sheep’s cheese was not reduced when rosemary essential oil was added to inhibit the growth of Clostridium tyrobutyricum . In a similar report, Gammariello et al. found that 13 extracts and essential oils of orange species, grapefruit, spring lemon, parsley, and lemon Boyajian did not affect the survival of lactic acid bacteria in Fior di Latte cheese, while decreasing the population of pathogenic bacteria . Furthermore, treating Argentinean cheese with 200 mg/kg of oregano oil had no influence on Lactococcus lactis, Lactobacillus bulgaricus, or Streptococcus thermophilus growth or acidifying activity, compared to the control .
The addition of 0.03% Mentha longifolia oil to Feta cheese resulted in the highest viability of Lacticaseibacillus casei at low pH, compared with the other treatments containing < 0.03%. Electron microscopy showed that essential oils caused no harm to L. casei . Other studies indicated that some plant extracts may improve starter activity. The total count of starter cultures (L. lactis ssp. lactis and L. lactis ssp. cremoris) was higher in the ginger-fortified UF-soft cheese and Egyptian white cheese pickled in a brine solution containing fresh ginger extract, compared to control cheese [38, 58]. Inversely, some studies indicate that some extracts have an adverse effect on the growth of starter cultures. For example, adding 2.5 µg/mL of thyme oil to Coalho cheese reduced the viable cell count of L. monocytogenes and the counts of starter cultures composed of L. lactis ssp. cremoris and L. lactis ssp. lactis .
Adding Cinnamom umverum and Allium sativum aqueous extracts to goat, cow, and camel milk had no significant effect on the acidification through fermentation . The aqueous extracts of rose flower, spearmint, dill, and green tea, as well as chamomile essence increased Bifidobacterium bifidum and L. acidophilus growth in probiotic milk and yoghurt and kept the bacteria alive until the end of storage, with no need for additional nutrients [61–63].
In another study, adding A. sativum or Cinnamomum verum water extracts to both cow and camel milk yoghurts boosted B. bifidum viability for 21 days of storage, compared to the control yoghurts. This was correlated to the presence of vitamins, minerals, amino acids, and polyphenolics in A. sativum and C. verum, among other factors. Camel milk has more free amino groups and a higher buffering capacity than cow milk, which leads to increased B. bifidum viability . Haddadin found that increasing concentrations of ethanol olive leaf extract accelerated bacteria growth and allowed them to attain optimal acidity in less time . The samples containing 0.6% of the extract had the highest bacteria count and the bacteria were viable until the end of fermentation. Polyphenols (oleuropein and other secoiridoids), flavonoids (rutin, flavonol), and luteolin-7-glucoside are stimulatory components in ethanol olive leaf extract.
In a different study, adding Diospyros kaki L. leaf extract to yoghurt increased the rate of acidification and decreased the time required to complete fermentation, contributing to the viability of the starter culture. In particular, the increase in the counts of S. thermophilus and Lactobacillus delbrueckii ssp. bulgaricus was the highest (2.95 and 1.14 log CFU/mL, respectively) in D. kaki yoghurt . The water cinnamon extract had no effect on the probiotic population, although Lactobacillus species and S. thermophilus counts in yoghurt increased for up to 7 days during storage .
Ziarno et al. showed that the herbal extracts from valerian, sage, chamomile, cistus, linden blossom, ribwort plantain, and marshmallow did not inhibit the growth of lactic acid bacteria in fermented milk, such as yoghurt . However, they can gradually inhibit fermentation at concentrations above 2% (w/w) and hence can be used to prevent post-acidification of fermented milk. In general, the plant extracts significantly increased the growth and acidification rates of S. thermophilus and L. delbrueckii ssp. bulgaricus. Plant extract components, including monosaccharides, formic acid, and hydroxycinnamic acid, as well as neochlorogenic, chlorogenic, and caffeic acids, play a stimulatory role and cause a beneficial effect on the growth of yoghurt culture bacteria through fermentation .
The viability of probiotic bacteria in fermented milk is also affected by the addition of essential oils. The yoghurts with mint, bee balm, and ziziphora essential oils (0.001%) exhibited higher viability of L. acidophilus LA-14 and Bifidobacterium animalis ssp. lactis BB-12 than the control samples. However, the yoghurt with eucalyptus essential oil had lower viability . The survivability of probiotic bacteria in yoghurt and cheese were unaffected by the essential oils of M. longifolia, Teucrium polium, Cuminum cyminum, Allium ascalonicum, and Pimpinella anisum . The viability of L. casei in a bio-yogurt containing various amounts of T. polium essential oil was significantly reduced after 28 days of storage. A probiotic yoghurt without essential oils and a bio-yogurt containing 60 ppm of T. polium oil had the highest overall viable count of L. casei (6.47 log CFU/mL). However, higher concentrations of the oil resulted in decreased bacterial counts . Sarabi-Jamab & Niazmand reported that the population of L. acidophilus in bio-yogurt with varied concentrations (25, 40, 70, 100, and 130 g/L) of Mentha piperita and Ziziphora clinopodioides essential oils considerably decreased after 7 days of storage . Yet, their viability did not change significantly, compared to the control. In ice cream, the probiotic bacteria L. acidophilus La-5 and B. bifidum Bb-12 were more stable during storage at –20°C for 90 days when made with tiger-nut extract, compared to the control .
Plant extracts as natural antioxidants. Mode of action. Antioxidants are essential for lowering oxidative reactions in food systems and the human body. In food systems, they retard lipid peroxidation and the formation of secondary lipid peroxidation products. Antioxidants also help to reduce protein oxidation, as the interaction of lipid-derived carbonyls with proteins alters their function . Dairy products contain lipids rich in polyunsaturated fatty acids and their esters are easily oxidized by molecular oxygen over time. This oxidation may occur during the manufacture, storage, or distribution of final products. Light, oxygen, and transition metal ions are important factors leading to oxidative changes. Deleterious changes in dairy products caused by lipid oxidation include not only offflavors but also the loss of color and nutrients, and the accumulation of compounds that may be detrimental to consumers’ health . Objectionable odors and flavors in oxidized products are caused by product subcomponents forming compounds such as hydrocarbons, aldehydes, and ketones .
Synthetic antioxidants are commonly used to increase the shelf life of food products, including TBHQ, BHA, and BHT. They reduce the rate of lipid oxidation and hydrolysis, as well as stabilize free radicals. According to the International Dairy Foods Association, these antioxidants are not allowed to be added to milk. Therefore, dairy products are fortified with natural antioxidants that delay lipid oxidation and hydrolysis, reduce nutritional losses, prevent free radical damage, and provide a variety of health benefits .
Antioxidants can be divided into primary and secondary based on how they work to reduce lipid oxidation. Primary antioxidants act as H donors to the lipid-free radicals formed during lipid oxidation and rearrangement into a stable form. Secondary antioxidants act as chelators of metal ions, decompose hydroperoxide into non-radical species, deactivate singlet oxygen, and absorb ultraviolet radiation. They can also act as oxygen scavengers to slow down the rate of radical formation .
Plant antioxidants can be divided into three categories: phenolic compounds, vitamins, and carotenoids . Phenolic compounds have a large diversity of structures: from simple molecules (e.g., ferulic, gallic, and caffeic acids) to polyphenols (tannins and flavonoids) . Vitamins E and C are the most important plant antioxidants. Vitamin E is a lipid-soluble vitamin made up of four tocopherols and four tocotrienols, each having four isomers (α, β, α, and δ), but only α-tocopherol can be absorbed by the human body. Vitamin C is a water-soluble vitamin found in a variety of fruits and vegetables . Therefore, these compounds have been considered promising candidates as potential protectors against lipid oxidation. The presence of an antioxidant is one of the fastest ways to reduce fat oxidation [80, 81].
Plant extracts as antioxidants in dairy products. Plant extracts containing high amounts of phenolic compounds act as H donors, radical scavengers, or metal chelators. Scientists have studied the antioxidant properties of sedge, marjoram, wild marjoram, caraway, basil extract, ginger, plum concentrates, aloe vera, mustard, tea catechins, rosemary extracts, and other plant extracts [82–84]. The antioxidant activity of milk increased significantly when plant extracts were introduced before bacterial fermentation .
Santos et al. used rosemary extract to prevent fat oxidation in cow milk fortified with fish oil. When tested at 60 and 110°C, an ethanol extract of rosemary added to 400 mg/kg of butter increased the butter’s oxidation stability . Plant extracts enhanced the antioxidant activity and overall phenolic content in the fermented milk. For example, marjoram extract added to yoghurt had a significant antioxidant effect on both the first and the last days of storage (28 days) . Cudrania tricuspidata L. and Morus alba L. leaf extracts improved yoghurt’s antioxidant activity and total phenolic content. C. tricuspidata leaf extract exhibited the highest antioxidant activity . Srivastava et al. found that the goat milk yoghurt fortified with 2% beet root or 2% ginger extracts had the highest antioxidant activity evaluated by the DPPH assay, followed by the cow milk yoghurt with 2% ginger extract . Furthermore, fortifying milk and yoghurt with 2% red ginseng extract increased its oxygen radical absorption capacity and radical-scavenging activity (DPPH), but reduced the DNA damage caused by H2O2, compared to the control yoghurt . The cheese fortified with clove extract was shown to have the strongest antioxidant and antibacterial qualities, compared to the cheeses fortified with other extracts (cinnamon stick, oregano, pomegranate peel, and grape seed) .
In recent years, herbal ghee has primarily been sold as medical ghee on the global market . This product has a typical flavor, a bitter or pungent aftertaste, and a dark color. The antioxidant activities of vidarikand (Pueraria tuberosa L.), shatavari (Asparagus racemosus L.), and ashwagandha (Withania somnifera L.) extracts were evaluated against the synthetic antioxidant BHA. Compared to the aqueous extracts, the herbs’ ethanol extracts were more effective in avoiding the formation of peroxide value and conjugated dienes in ghee. Puerarin, daidzein, genistein, and daidzin are active ingredients in vidarikand .
Parmar et al. discovered that a 7% ethanol extract of Terminalia arjuna L. bark was particularly effective in preventing auto-oxidation of both cow and buffalo ghee during storage. The extract had a substantial ability to increase the antioxidant potential of ghee, with the efficacy being greater in cow ghee than in buffalo ghee. The Arjuna-fortified ghee had a shelf life of 8 days at 80 ± 1°C, compared to only 2 days for the control ghee sample .
Sage and rosemary extracts have been the most widely used herbs to prolong the shelf life of ghee and butter oil . These extracts have many times the antioxidant activity of synthetic antioxidants like BHA and BHT . Butter oil supplemented with dihydroquercetin (DHQ) as a natural antioxidant showed the strongest oxidative stability in the accelerated test. The addition of 50, 100, 150, and 200 ppm of DHQ increased the shelf life of butter oil by 1.9, 2.8, 2.99, and 3.53 times, respectively . Similarly, adding 80 mg of olive mill waste water or pomace to 1 kg of butter provided oxidative stress resistance during storage both under ambient thermal conditions (25°C) and the oven conditions (60°C) for three months . During storage, the butter made from sour cream supplemented with 2% sage or rosemary had higher oxidative stability and lower secondary oxidative products, including malonaldehyde and ketones, than the control butter. However, rosemary herb was found to be more efficient than sage in slowing lipolysis in butter .
Similarly, Merai et al. found that the ghee made from butter and 0.6% Tulsi (Ocimum sanctum L.) leaves extract was as stable as the ghee containing 0.02% of BHA after 8 days of high temperature storage (80 ± 2°C) . The phenolics found in Tulsi leaves appeared to be the primary contributors to ghee’s increased oxidative stability. Furthermore, Farag et al. reported that adding thyme and cumin essential oils to butter prevented it from spoiling at room temperature and was more efficient than butylated hydroxy toluene .
Plant extracts as natural milk-clotting enzymes. The global increase in cheese production, along with a decreased supply and higher prices of calf rennet, has led to the search for alternative milk-clotting enzymes as suitable rennet substitutes. Plant clotting enzymes, also known as plant proteases, have become a subject of growing interest in the cheese industry due to their availability, simple purification processes, and low cost, as well as stability [99, 100]. The selection of a suitable plant coagulant depends on the optimum conditions for enzyme activity (pH, temperature, salt, solvents, etc.), milk-clotting activity/proteolytic activity (MCA/PA ratio), and the rheological and sensory properties of final products .
Plant proteases. In general, the main classes of milkclotting proteases are aspartic, serine, and cysteine proteases. The number and type of enzymes vary from one species to another and depend on the plant parts . As chymosin, some plant proteases can cleave a few sites at αs1- and β-caseins, which may occur in maintaining the micelle stability. These regions of αs1- and β-caseins are sometimes near the micelle surface and contribute to electrostatic repulsion between casein micelles. The removal of these parts could greatly assist the gelling process. First, the initial instability of micelles is increased and coagulant access to β-casein is improved. Second, the removal of these parts increases the flexibility and/or susceptibility of caseins to rearrangements in gel .
Plant proteases also play a significant role in the early stages of cheese ripening. The hydrolysis of caseins in cheese by residual coagulants produces essential substrates for some bacterial microflora, whose breakdown allows for flavor development during maturation. The strength of these impacts on the cheese quality depends on the type of plant coagulant used, its amount, and its enzymatic activities . Some plant milk-clotting proteases are presented in Table 2.
Several studies have reported that most plant proteases with milk-clotting activity (MCA) are stable in various pH ranges (4.5–10) and temperatures (20–80°C), with the optimum pH around 6.5 and maximum activity around 60°C. The protease isolated from pumpkin seed extract curdled milk at a pH range of 4.5 to 8.5 and a temperature range of 20 to 80°C. It was resistant to solvents, salts, and surfactants, and was more effective on κ-casein than β-casein . The best coagulation conditions for the pineapple, kiwi, and ginger extracts were pH 5, 6.6, and 6.6, respectively, and temperatures 45, 40, and 45°C, respectively . A novel cysteine protease extracted from Ficus johannis L. by cation exchange chromatography was stable in a variety of pH ranges (3.0–10.5), with the optimum at 6.5, and showed maximal activity at 60°C. The purified protease had significant activity against κ-casein when compared to α- and β-casein. In the presence of high salt concentrations, the enzyme was virtually totally active . Ben Amira et al. showed that when the pH was dropped to 3, the MCA/PA ratio rose, surpassing that of chymosin. The lowest ratio attributed to the extract at pH 6 was mainly related to its high proteolytic activity, as well as to its low MCA. Melon extracts also showed high milk-clotting activity over a wide range of temperatures (45–75°C), while kiwi and ginger extracts showed high activity over a lower temperature range, with a maximum of 40 and 63°C, respectively .
Effect of plant proteases on yield and physiochemical properties of cheese. The rheological properties of milk gels and sensory characteristics of cheeses produced by plant proteases vary according to the type of coagulant, its enzymatic activities, and its concentrations. Most plant enzymes are not suitable for industrial-scale cheese production, where a large portion of protein is lost due to excessive hydrolytic activities . For example, the curd yield produced using kiwi (17.8%), melon (15.1%), and ginger (15.4%) extracts was lower than that produced using commercial rennet (20.2%). Kiwi extracts had textural properties comparable with those obtained using calf rennet, thus holding the best potential as a milk coagulant in cheese production. Melon extracts, however, produced a fragile gel and a low curd yield . The cheese made with the purified enzyme obtained from F. johannis had similar textural properties and chemical compositions to the cheese produced using commercial calf rennet . All rheological parameters indicated a strong milk gel formed using pumpkin seed extracts. The peptidase sequence was homologous with that of cucumicin-like peptidase [99, 105].
Abebe & Emire used Calotropis procera L. enzyme leaf extract as an alternate milk coagulant to produce fresh cheese . The highest cheese yield and the fastest clotting time were 17.89 kg of cheese/100 kg of milk and 14:50 min, acquired with 10 g of C. procera powder at 60°C extraction, respectively. There was no difference in coagulation time among the milk samples with varied milk fat structures. However, whey was extracted from homogeneous cream curds more quickly than from non-homogenized cream curds . Camel milk is difficult to convert to cheese using regular rennet, so pineapple, kiwi, and ginger enzyme extracts are used to help make cheese from camel milk. Kiwi extract showed the highest curd yield (20.71%) when compared to pineapple (19.74%) and ginger (11.50%) extracts . Mazorra-Manzano et al. reported that kiwi had a higher output than melon and ginger but was lower than chymosin in cow milk . When compared to the camel cheese with pineapple and ginger extracts, the camel cheese with kiwi extract had higher amounts of water-soluble vitamins, primarily B7 (3.75034 mg/g), B12, and B5, as well as higher mineral contents, primarily Na (605.2 ppm) and Ca (63.11 ppm). The water content was lowest in the camel milk cheese made with ginger extract, whereas the protein content was higher in the cheese made with pineapple extract than kiwi or ginger extracts . Gad & Abd El-Salam mentioned that higher concentrations of rosemary extract increased the rennet coagulation time of skim milk. The antioxidant activity of the skim milk/rosemary extract blends was improved by heat treatment, calcium chloride addition, and pasteurization . Furthermore, by suppressing oxidation, rosemary extract as a natural antioxidant could extend the shelf life of Cheddar cheese or cheese powder .
Plant extracts and sensory properties of dairy products. Flavor, an important sensory component, is one of the variables influencing customer preferences. The flavor of food is determined by the presence of volatile aromatic compounds, which take different forms in different foods . Flavoring compounds can be roughly classified into plant-based, artificial, and biotechnologically formed flavors. Plant-based flavors are separated from plant-based sources rich in aromatic compounds, spices like vanilla, or herbs. Modupalli et al. reported that plant-based food flavoring agents are naturally occurring polyphenolic compounds, organic esters, acids, alkaloids, and carotenoids . Dry aromatic plants, as well as their essential oils and extracts, are used in dairy products in order to give them a distinctive and attractive taste and smell . In cheese, the ethanol cinnamon extract improved the flavor and overall quality of flavored processed cheese, whereas lemon grass and cress extracts improved its odor and color, respectively . The UF-soft cheese produced with the ethanol ginger extract became more pleasant compared to the control cheese, especially during storage . The major compounds responsible for the unique ginger flavor are gingerols and other volatile oils. Over storage, gingerols are converted into a series of homologous compounds known as shogaols, which are more pungent than gingerols . Mahajan et al. improved the flavor, texture, and acceptability of low-fat Kalari cheese by using aqueous pine needle extracts . Similarly, the aqueous extracts of Inula britannica L. increased the odor and flavor of a Cheddar-type cheese . The cottage cheese with 8 and 9% aqueous green tea extract acquired a pleasant, moderately expressed green tea flavor and taste, whereas the cottage cheese with a high level of tea extract (> 9%) had a bitter and disagreeable tea flavor [116, 117].
Essential oils improve the flavor and smell of dairy products and also extend their shelf life. The highest overall acceptability during storage was achieved by Iranian white cheese containing 0.75% basil oil, followed by the sample containing 0.5% salvia oil. However, adding 0.75 and 1% salvia oil impaired the samples’ odor and taste . Iranian white cheese and Domiati cheese were also the most preferable and had the highest flavor scores when supplemented with 1 and 0.2% black cumin essential oil [38, 41]. Inversely, the goat cheeses treated with essential oils had a bitter flavor, whereas Feta cheese had a strong off-taste due to large quantities of clove and tea tree oils required for antibacterial activity . Furthermore, Foda et al. observed that high concentrations of spearmint oil can generate concerns about changes in white cheese’s sensory properties, to the point that the panel test revealed the highest acceptability at lower oil concentrations .
In fermented milk, adding the extracts of D. kaki leaf and Nelumbo nucifera L. leaf to yoghurt increased its viscosity, water-holding capacity, bitterness, and texture smoothness. The D. kaki – fortified yoghurt contained a high content of flavoring components such as acetaldehyde, acetoin, and diacetyl, which gave it the best taste in the sensory evaluation. The N. nuciferafortified yoghurt showed the largest amount of acetic acid in the volatile complex analysis and the highest pH value . According to Zaky et al., adding 2 μL/100 mL of dill and caraway essential oils to Labneh made from buffalo’s milk increased the total volatile fatty acids of Labneh during storage . In addition, it improved its antioxidant activity and sensory qualities, compared to the control. Ghalem & Zouaoui added Rosmarinus officinalis L. oil to yoghurt at concentrations of 0.14, 0.21, 0.29, and 0.36 g/L and stored it for up to 21 days . The herbal yoghurt enhanced with 0.14 g/L of the oil received the highest score for taste, flavor, and texture from the panel. Trivedi et al. tested basil in various forms (juice and dried powder) as an ice cream flavoring agent. Compared to the control, adding basil juice (up to 2%) lowered protein, fat, total solids, ash, and total carbohydrate contents, as well as melting resistance and pH . Coffee extract was also used to produce a distinct probiotic coffee ice cream with the desired coffee flavor that improves the consumer’s emotional state and aids in calorie burning . Table 3 represents some dairy products fortified with flavoring plant extracts and their bioactive components.
Encapsulated plant extracts in dairy products. Encapsulation is a process of entrapping one substance into another to improve the bioavailability of high-value compounds . It can be used to mask undesirable flavors and odors, protect biologically active compounds from adverse interactions with other substances, and improve stability under a variety of environmental conditions, including temperature, moisture, oxidation, and light . Salama et al. summarized numerous forms for encapsulating bioactive compounds, including nanocapsules, micro-capsules, nano-emulsions, micro-emulsions, solid lipid nanoparticles, liposomes, and others. The nano- or micro-capsules are excellent systems that deliver bioactive compounds for direct absorption. Dairy products fortified with encapsulated plant extracts have better nutrition and health benefits. There is a variety of materials used for encapsulation, including gum Arabic, modified starches, maltodextrins, alginates, pectin, carrageenan, hydrogenated vegetable oils, bees wax, soy proteins, gelatins, whey proteins, sodium caseinates, and others. Below are some examples of recent and most common applications of encapsulation technology in dairy products .
The encapsulation of sage extract in liposome improved its antimicrobial activity against pathogenic bacteria and affected the physicochemical properties of the resulting yoghurt. Particularly, acidity increased, while diacetyl and acetaldehyde decreased when the extract was added at rates of 5, 10, 15, and 20%. The yoghurt’s viscosity first increased but then decreased during storage . The low antioxidant activity in dairy products was corrected by fortifying them with doum fruit extract in a liposome form. The encapsulated doum extract has a significant impact on yoghurt’s chemical analysis, especially at higher concentrations . El-Messery et al. investigated the encapsulation of mango peel phenolic extract and its use in milk beverages . They found that these flavored drinks were well-liked by consumers. They were high in antioxidants, phenolic compounds, and many other bioactive components. In addition, the encapsulated extract did not alter the chemical or rheological aspects of the beverage. In another study, chitosan microcapsules containing beetroot or ginger aqueous extracts were added to fermented camel milk in the presence of probiotic bacteria. The 10% concentration of the beetroot aqueous extract microcapsules increased the survivability of probiotic bacteria, whereas the amount of ginger was only 1%. The best effects were achieved by the chitosan beads with the beetroot aqueous extract . Jaboticaba-loaded nanoemulsion (up to 15%) was added to cow milk to make it rich in phenolics and exhibit high antioxidant activity . Adding the fig leaf extract microencapsulated with alginate and/or skim milk improved the cheese sauce’s properties, microbiological quality, and shelf life . The control cheese and the functional cheese supplemented with 2% liposomal encapsulated saffron extract showed the greatest difference in terms of chemical composition and color. The cheeses containing the encapsulated saffron extract were significantly harder and chewier, compared to the control. However there was no significant difference in adhesiveness, cohesiveness, and gumminess among the cheese samples. Based on the findings, liposomal encapsulation was considered an efficient method for the delivery of saffron extract to ricotta cheese as a novel functional food .
In another study, the encapsulated Arjuna herb extract improved the vanilla chocolate dairy drink in all the parameters tested by the response surface methodology . Barretto et al. reported that the encapsulated anthocyanin provided yoghurt with high stability and large amounts of antioxidant and phenolic compounds. Encapsulation gave anthocyanin more efficiency and biological activity . Sawale et al. studied the effect of heat treatment on the stability of the T. arjuna extract and its content of phenols and antioxidants when it was used in the free and encapsulated forms to fortify the vanilla chocolateflavored milk drinks . They found that sterilization had a negative effect on the extract in the free form and its content of antioxidants and phenols. The encapsulated extract, however, was stable enough to protect the bioactive compounds. Lourenço et al. studied the encapsulation of pineapple peel hydroalcoholic extract rich in phenolic compounds. To preserve these ingredients, maltodextrin, inulin, and Arabic gum were used as encapsulation materials by spray drying. The resulting powder had a good flow ability and suitable handling properties. Also, this encapsulation method ensured high antioxidant activity, while the non-encapsulated extract had low activity in the same conditions .
ВЫВОДЫAs compared to plants or their parts, the use of plant extracts and essential oils in the dairy industry has received a lot of positive feedback from both dairy producers and consumers. Plant extracts can be used to enhance the flavors, antioxidant and antibacterial properties of fortified dairy foods, as well as their visual appeal. Additionally, plant extracts can be added to dairy products in a variety of forms, free or encapsulated, to protect active ingredients from the external environment and control their release. They can also be incorporated as a nanoemulsion to enhance the compounds’ bioactivity. Plant extracts and their essential oils have different effects on microorganisms, depending on the type of microbe, the type and concentration of the plant extract, and the interaction of antimicrobial chemicals with the food matrix. Most studies indicate that plant extracts and essential oils included in various dairy products increase the survival of probiotics and starter bacteria while acting as antimicrobials against pathogenic and spoilagecausing microorganisms. With the growing demand for cheese in the world, plant-based coagulants can be a good substitute for calf rennet, which is currently in low supply. Different plant-derived proteases can be used in milk coagulation and cheese-making. However, to produce cheeses that are comparable to those made with calf rennet, we need to select the right type of plant proteases (specific or non-specific action) and their enzymatic activity (milk-clotting activity/proteolytic activity ratio), depending on the type of cheese to be produced (ripened or non-ripened). In the dairy industry, plant extracts can also be used to either increase desirable ingredients (vitamins, fiber, and minerals) or to partially replace undesirable ingredients (salt and sugar).
Вклад авторовThe authors were equally involved in writing the manuscript and are equally responsible for plagiarism.
КОНФЛИКТ ИНТЕРЕСОВThe authors state that there is no conflict of interest.
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