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Impact of transglutaminase treated micellar casein concentrate and milk protein concentrate on the functionality of processed cheese product slice formulations

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  • Milk protein concentrate (MPC) is used as an ingredient in processed cheese product (PCP) formulations. However, its use can result in texture defects such as a soft body and restricted melting characteristics. The use of micellar casein concentrate (MCC), which has a higher level of casein and less serum protein, improves the texture of PCP. Further improvement in PCP products may be possible using transglutaminase (TGase), an enzyme that can crosslink proteins. This study aimed to determine the effect of TGase treatment of MPC and MCC retentates on the functionality of MPC and MCC when used in a PCP slice formulation. Three lots of MCC and MPC retentate were produced using microfiltration and ultrafiltration, respectively. Each replicate of retentate was divided into three equal portions and treated with transglutaminase enzyme at three different levels: 0.3 U/g of protein, 3.0 U/g of protein, and no TGase addition. The retentates were spray-dried, and powders were used in PCP slice formulation. Functional properties of PCP were analyzed using a penetration test, Dynamic stress rheology (DSR) for transition temperature (TT), and Schreiber melt test. As the TGase addition increased, there was a significant (p ≤ 0.05) increase in TT and a significant (p ≤ 0.05) decrease in the Schreiber melt area. The PCP made from MCC had higher TT and Schreiber melt area values than that made from MPC as an ingredient (TGase or no TGase). It was concluded that TGase treatment modifies the melt characteristics of MCC and MPC in PCP applications.
  • Bletilla Rchb. f. is one of the most economically valuable groups of orchids in the world. Due to its ornamental significance, the genus Bletilla occupies an important place in the worldwide horticultural market. Furthermore, in China, Japan, South Korea, and other Asian countries, it is highly valued for its medicinal use[1].

    There are eight species in the genus Bletilla, including Bletilla chartacea (King & Pantl.) Tang & F.T. Wang, Bletilla cotoensis Schltr., Bletilla foliosa (King & Pantl.) Tang & F.T. Wang, Bletilla formosana Schltr., Bletilla guizhouensis J. Huang & G.Z. Chen, Bletilla morrisonensis Schltr., Bletilla ochracea Schltr., and Bletilla striata Rchb.f.[2,3]. The distribution area spans from northern Myanmar in Asia to Japan via China[4]. Five species are native to China, namely, B. foliosa, B. formosana, B. guizhouensis, B. ochracea, and B. striata. In China, people have assigned various names to Bletilla based on its morphology and efficacy, such as baiji (白及/白芨), baigen (白根), baige (白给), baijier (白鸡儿), baijiwa (白鸡娃), diluosi (地螺丝), gangen (甘根), junkouyao (皲口药), lianjicao (连及草), and yangjiaoqi (羊角七)[5]. These diverse appellations highlight the importance of this genus in Chinese folk biological culture.

    The medicinal material known as 'baiji' in traditional Chinese medicine (TCM) is usually the dried tuber of B. striata, which is also the authentic product included in the Chinese Pharmacopoeia[6]. According to the Chinese Pharmacopoeia (2020), TCM baiji is sliced, dried, and crushed into a powder that can be used topically or internally, with a recommended dosage of 3–6 g at a time, offering astringent, hemostatic, detumescence, and myogenic effects. It is often used for conditions such as hemoptysis, hematemesis, traumatic bleeding, sores, and skin chaps[7]. Although only B. striata is the authentic product of TCM baiji, the other four Bletilla species native to China are also used as substitutes, and this practice is widespread[8].

    Modern research indicates that Bletilla contains a variety of chemical components, including benzol, dihydrophenanthrene, phenanthrene, and quinone derivatives. These components confer pharmacological effects on Bletilla, such as hemostasis, anti-tumor activity, and promotion of cell growth[9]. Due to its outstanding medicinal value, Bletilla can be found in nearly every corner of the traditional medicine market (Fig. 1). However, habitat destruction and uncontrolled mining have led to a significant reduction in the native populations of Bletilla, making its protection an urgent priority. Therefore, this paper provides a comprehensive review of relevant research up to August 2023, covering botanical characteristics, resource distribution, ethnobotanical uses, chemical components, pharmacological effects, clinical applications, and safety evaluations of Bletilla. The aim is to raise awareness and promote the protection and sustainable use of this genus.

    Figure 1.  Varieties of Bletilla at the traditional March Medicinal Market in Dali, Yunnan, China.

    The morphology of different Bletilla species is highly similar. The primary taxonomic feature distinguishing each species is the characteristics of the flower, particularly the lip of the flower, including its size, shape, and the number and shape of longitudinal ridges on the lip plate (Table 1, Fig. 2)[1014].

    Table 1.  The morphological differences among five species of Bletilla plants native to China.
    Morphological featureBletilla striataBletilla formosanaBletilla ochraceaBletilla foliosaBletilla guizhouensis
    Plant height (cm)18−6015−8025−5515−2045−60
    Rhizome shapeCompressedCompressedSomewhat compressedSubgloboseCompressed
    Rhizome diameter (cm)1−31−2About 21−1.53−4
    Stem characteristicsStoutEnclosed by sheathsStoutStout, shortThin
    Leaf shapeNarrowly oblongLinear-lanceolateOblong-lanceolateElliptic-lanceolateNarrowly lanceolate
    Leaf size (cm)8−29 × 1.5−46−40 × 0.5−4.58−35 × 1.5−2.85−12 × 0.8−325−45 × 1.2−4.5
    Flower colorPurplish red or pinkPale purple or pinkYellowPale purpleDeep purple
    Flower sizeLargeMediumMediumSmall to mediumLarge
    Inflorescence structureBranched or simpleBranched or simpleSimpleSimpleBranched
    Pedicel and ovary length (mm)10−248−12About 187−913−17
    Sepal shapeNarrowly oblongLanceolateLanceolateLinear-lanceolateOblong-elliptic
    Petal shapeSlightly larger than sepalsSlightly narrower than sepalsObliqueLanceolateOblong-elliptic
    Lip shapeObovate-ellipticBroadly ellipticNarrowly rhombic-obovateNarrowly oblongNarrowly oblong
    Lip colorWhite with purplish veinsWhitish to pale yellow with small dark purple spotsWhitish to pale yellow with small dark purple spotsWhite with purplish spots and purple edgeWhite with deep purple edge
    Number of lip Lamellae5 lamellae5 undulate lamellae5 longitudinal lamellae3 fimbriate lamellae7 longitudinal lamellae
    Column characteristicsSubterete, dilated towards apexSubterete, dilated towards apexSlender, dilated towards apexCylindric, dilated towards apexSuberect, with narrow wings
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    Figure 2.  (a)−(d) Bletilla striata (Thunb. ex Murray) Rchb. f. (e)−(h) Bletilla formosana (Hayata) Schltr. (i)−(l) Bletilla ochracea Schltr. (m), (n) Bletilla sinensis (Rolf) Schltr. (o), (p) Bletilla guizhouensis Jie Huang & G.Z. Chen (Photographed by Wang Meina, Zhu Xinxin, and He Songhua).

    The flowers of B. striata are large and purplish-red or pink, with narrowly oblong sepals and petals measuring 25−30 mm in length and 6−8 mm in width. They have acute apices, nearly as long as the sepals and petals. The lip is obovate or elliptic, predominantly white with purplish-red coloration and purple veins, measuring 23−28 mm in length, slightly shorter than the sepals and petals. The lip disc exhibits five longitudinal folds extending from the base to near the apex of the middle lobe, with waviness occurring only above the middle lobe[11]. In China, B. striata is found in regions such as Anhui, Fujian, Guangdong, Guangxi, Gansu, Guizhou, Hubei, Hunan, Jiangsu, Jiangxi, Shaanxi, Sichuan, and Zhejiang. It also occurs in the Korean Peninsula and Japan, thriving in evergreen broad-leaved forests, coniferous forests, roadside grassy areas, or rock crevices, at altitudes ranging from 100−3,200 m[12].

    B. ochracea's flowers are medium to large, featuring yellow or yellow-green exteriors on the sepals and petals, while the insides are yellow-white, occasionally nearly white. The sepals and petals are nearly equal in length, oblong, measuring 18−23 mm long and 5−7 mm wide, with obtuse or slightly pointed apices, often adorned with fine purple spots on the reverse side. The lip is elliptic, typically white or light yellow, measuring 15−20 mm in length and 8−12 mm in width, with three lobes above the middle. The lip disc is characterized by five longitudinally ridged pleats, with undulations primarily occurring above the middle lobe[13]. B. ochracea is native to southeastern Gansu, southern Shaanxi, Henan, Hubei, Hunan, Guangxi, Guizhou, Sichuan, and Yunnan, thriving in evergreen broad-leaved forests, coniferous forests, or beneath shrubs, in grassy areas or alongside ditches at altitudes ranging from 300−2,350 m[14].

    B. formosana's flowers come in shades of lavender or pink, occasionally white, and are relatively small. The sepals and petals are narrowly oblong, measuring 15−21 mm in length and 4−6.5 mm in width, and are nearly equal in size. The sepals have subacute apices, while the petal apices are slightly obtuse. The lip is elliptic, measuring 15−18 mm in length and 8−9 mm in width, with three lobes above the middle. The lip disc exhibits five longitudinal ridge-like pleats, which are wavy from the base to the top of the middle lobe[15]. B. formosana is indigenous to southern Shaanxi, southeastern Gansu, Jiangxi, Taiwan, Guangxi, Sichuan, Guizhou, central to northwest Yunnan, southeast Tibet (Chayu), and Japan. It is typically found in evergreen broad-leaved forests, coniferous forests, road verges, valley grasslands, grassy slopes, and rock crevices, at altitudes ranging from 600−3,100 m[16].

    The flowers of B. foliosa are small and lavender, with white sepals and petals featuring purple apices. The sepals are linear-lanceolate, measuring 11−13 mm in length and 3 mm in width, with subacute apices. The petals are lanceolate, also measuring 11−13 mm in length and 3 mm in width, with acute apices. The lip is white, oblong, adorned with fine spots, and features a purple apex. It measures 11−13 mm in length and 5−6 mm in width, tapering near the base and forming a scaphoid shape. The lip is anteriorly attenuated, unlobed, or abruptly narrowing with inconspicuous three lobes and exhibits fringe-like fine serrations along the edge. Three longitudinal ridge-like pleats are present on the upper lip disc[17]. B. foliosa typically grows on hillside forests, with its type specimen collected from Mengzi City, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province, China[17].

    B. guizhouensis is a recently discovered species in Guizhou, China. In terms of shape, B. guizhouensis closely resembles B. striata, but it can be distinguished by its ovate-oblong buds, oblong dorsal sepals, obovate lips, and middle lobes of the lips, which are oval in shape. The disc of B. guizhouensis features seven distinct longitudinal lamellae, setting it apart from other known Bletilla species and establishing it as a distinct species[2]. Presently, B. guizhouensis has only been found in Guizhou, China, primarily thriving in evergreen broad-leaved forests at altitudes ranging from 900−1,200 m[3].

    Understanding the morphology, habitat, and distribution of Bletilla species is crucial for the conservation and propagation of these resources. To effectively implement plant conservation and breeding programs, a comprehensive understanding of the specific morphological characteristics, growth environments, and native habitats of these plants is essential, as without this knowledge, effective results cannot be achieved.

    The ethnobotanical uses of Bletilla worldwide primarily fall into two categories: ornamental and medicinal purposes. Bletilla orchids, renowned for their striking and distinct flowers, are commonly cultivated for ornamental purposes across many countries[18]. Valued for their aesthetic appeal, these orchids are frequently grown in gardens and utilized as potted plants. Among the various cultivars, B. striata stands out as the most favored choice for ornamental horticulture due to its ease of cultivation and adaptability to diverse climates[19,20].

    Contrastingly, in select Asian countries, Bletilla assumes a crucial role as a medicinal plant. For instance, influenced by TCM, the tuber of Bletilla also serves as a crude drug for hemostatic and anti-swelling purposes in Japan[21]. Likewise, traditional Korean medicine, deeply rooted in TCM principles, extensively documents the versatile use of Bletilla in addressing issues such as alimentary canal mucosal damage, ulcers, bleeding, bruises, and burns[22]. In Vietnam, Bletilla has been used as a medicinal herb for treating tumors and skin fissures, aligning with practices observed in the ethnic communities of southwest China[23].

    In China, Bletilla boasts a longstanding medicinal history, with numerous classical ancient Chinese medicine books containing detailed records of its medicinal applications[2432]. Even in contemporary society, many ethnic groups residing in mountainous areas in China continue to uphold the traditional medical practice of using Bletilla medicinally[31].

    In ancient Chinese medical literature, detailed records of Bletilla's morphology can be traced back to the late Han Dynasty, around 200 AD[24]. The Mingyi Bielu, a historical source, documented, 'Bletilla grows in the valley, with leaves resembling those of Veratrum nigrum L., and its root is white and interconnected. The ideal time for harvesting is September'. As awareness of the medicinal significance of Bletilla grew, successive dynastic-era Chinese medical texts consistently included descriptions of Bletilla's morphology (Table 2). In the Ming Dynasty, Li Shizhen compiled these earlier accounts of Bletilla's plant characteristics in his work, the Compendium of Materia Medica. He even provided an illustrative depiction of this plant genus (Fig. 3)[25].

    Table 2.  Morphological description of the plants belonging to Bletilla in the ancientChinese medicinal books.
    Dynasty (Year)TitleAuthorOriginal ChineseEnglish translation
    Late Han
    (184−220 AD)
    Mingyi Bielu/白给生山谷, 叶如藜芦,
    根白相连, 九月采
    Bletilla grows in the valley, with leaves like Veratrum nigrum L., root is white and connected. September is the time for harvesting.
    Wei-Jin period
    (220−420 AD)
    WuPu BencaoWu Pu白根, 茎叶如生姜, 藜芦,
    十月花, 直上, 紫赤色,
    根白连, 二月, 八月, 九月采
    Bletilla, stems and leaves like Zingiber officinale Roscoe and V. nigrum. It blooms in October and is purple and red, the inflorescence is vertical and upward. The roots are white and connected. It can be dug in February, August, and September.
    the Northern and Southern
    (420−589 AD)
    Bencao JizhuTao Hongjing近道处处有之, 叶似杜若,
    根形似菱米, 节间有毛
    It is everywhere near the road. The leaves are like Pollia japonica Thunb. The roots are like the fruit of Trapa natans L., and internode are many fibrous roots.
    Tang
    (618−907 AD)
    Su Jing, Zhangsun Wuji, etcTang materia medica生山谷, 如藜芦, 根白连, 九月采Born in the valley, with leaves like V. nigrum, root is white and connected. September is the time for harvesting.
    Song
    (960−1279 AD)
    Su SongCommentaries on the Illustrations白芨, 生石山上。春生苗,
    长一尺许, 似栟榈及藜芦,
    茎端生一台, 叶两指大, 青色,
    夏开花紫, 七月结实, 至熟黄黑色。
    至冬叶凋。根似菱米, 有三角白色, 角端生芽。二月, 七月采根
    Bletilla grow on the stone hill. It sprouts in spring and grows about a foot long. The seedlings are like Trachycarpus fortunei (Hook.) H. Wendl. and V. nigrum. The leaves are two finger-size. In summer, it blooms purple flowers and bears fruit in July. The ripe fruit is yellow-black. The leaves wither in winter. The root is like the fruit of T. natans, with three corners, white, and sprouting at the corners. The roots are dug in February and July.
    Ming
    (1368−1644 AD)
    Li ShizhenCompendium of Materia Medica一棵只抽一茎, 开花长寸许,
    红紫色, 中心如舌, 其根如菱米,
    有脐, 如凫茈之脐,
    又如扁扁螺旋纹, 性难干
    Only one stem per herb. The flower is more than one inch long, red and purple, and the center resembles a tongue. Its root is similar to the fruit of T. natans, possessing an umbilicus akin to that of Eleocharis dulcis (N. L. Burman) Trinius ex Henschel. It has spiral veins and is challenging to dry.
    −, Anonymous.
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    Figure 3.  Bletilla in Compendium of Materia Medica.

    Generally, ancient Chinese medical texts did not make clear distinctions between different Bletilla species. They collectively referred to plants with similar morphological traits as 'baige', 'baigen', 'baiji', 'gangen', 'lianjicao', or 'ruolan'. However, through textual analysis, it has been established that the descriptions of Bletilla in ancient texts before the Ming Dynasty largely align with Bletilla striata in terms of plant height, pseudo-bulb shape, leaf morphology, flower and fruit colors, and other characteristics. While the Bletilla portrayed in attached images may not precisely match B. striata in terms of morphology, considering the textual descriptions, it generally corresponds with B. striata. In writings from the Ming Dynasty and later periods, more specific descriptions of Bletilla emerged, encompassing details about its vascular arrangement, inflorescence, and flower structure, which consistently align with B. striata. Consequently, researchers have corroborated that the original plant of Bletilla described in ancient texts is Bletilla striata[24,33].

    According to the ancient Chinese medicinal books, Bletilla was used to treat a wide variety of conditions, including coughing, bruising, and bleeding, but their most mentioned use in ancient Chinese texts is for skin whitening and freckle removal[25]. Since ancient times, Bletilla species have been used consistently for skin care and whitening, and there are many well-known skincare products related to Bletilla. These Chinese formulae with Bletilla are similar to modern facial masks, face creams, facial cleanser, hand creams and other skin care products[26].

    For example, a prescription for 'facial fat (面脂)' in Medical Secrets from the Royal Library (752 AD) is made by boiling Bletilla with other traditional ingredients, and is applied to the face, resulting in skin whitening, freckle and wrinkle removal[27]. The 'Angelica dahurica cream (白芷膏)' in the General Medical Collection of Royal Benevolence (1111−1125 AD) is reputed to whiten facial skin through a seven-day treatment regiment, and contains Bletilla as the main botanical ingredient along with Angelica dahurica[28]. Jingyue Quanshu (1563-1640 AD) also contains a prescription called 'Yurong powder (玉容散)' for facial skin care. 'Yurong powder' is made of a fine powder of Bletilla, Nardostachys jatamansi (D. Don) DC., Anthoxanthum nitens (Weber) Y. Schouten & Veldkamp and other herbs[29]. Washing the face with Yurong powder in the morning and evening every day is said to make a person's face white without blemishes (Fig. 4)[29].

    Figure 4.  Yurong powder made of Bletilla and other traditional Chinese medicines in Jingyue Quanshu.

    In addition, in ancient Chinese medicine texts, Bletilla is also a well-known medicine for treating hematemesis, hemoptysis and bruises[23]. According to Shennong's Classic of Materia Medica (25−220), grinding the white fungus into fine powder and taking it after mixing with rice soup can be effective for treating lung damage and hematemesis[30]. Among the Prescriptions for Universal Relief (1406), 18 traditional Chinese medicines, such as Bletilla, are used to make 'snake with raw meat cream', which is said to be useful to treat carbuncles and incised wounds[31]. There is also a record of Bletilla powder treating lung heat and hematemesis in the Collected Statements on the Herbal Foundation (1624)[32].

    In ancient Chinese medicinal texts, most Bletilla are said to be useful for lung injury and hemoptysis, epistaxis, metal-inflicted wounds, carbuncles, burns, chapped hands and feet, whitening and especially for skin care. In the ancient medicinal texts, Bletilla is used alone or mixed with other traditional Chinese medicines. It is usually used in the form of a powder. The various medicinal effects of Bletilla described in these ancient texts suggest the great potential of this genus in clinical application, especially in the market of skin care products and cosmetics.

    As a skin care herb praised by ancient medical classics, 11 ethnic minorities in China, such as Bai, Dai, De'ang, Jingpo, Lisu, Miao, Mongolian, Mulao, Tu, Wa, and Yi still retain the traditional habit of using Bletilla for skin care in their daily life (Table 3). In addition to B. striata, B. formosana and B. ochracea are also used as substitutes. Although Chinese ethnic groups have different names for Bletilla spp., the skin care methods are basically the same. Dry Bletilla tubers are ground into a powder and applied to the skin[34], and this usage is also confirmed by the records in ancient medical texts[23, 24]. The various local names of Bletilla by different ethnic groups also indirectly suggests which ethnic groups play an important role in the traditional use. For example, Bai people called B. striata baijier (白鸡儿), goubaiyou (狗白尤), and yangjiaoqi (羊角七) (Table 3).

    Table 3.  The traditional medicinal knowledge of Bletilla in ethnic communities, China.
    Ethnic groupLatin nameLocal nameUsed partUse methodMedicinal effect
    AchangBletilla striata (Thunb. ex Murray) Rchb. F.BaijiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationTuberculosis, hemoptysis, bleeding from gastric ulcer, burns and scalds
    BaiBaijier, Goubaiyou, YangjiaoqiTuberTreatment of tuberculosis hemoptysis, bronchiectasis hemoptysis, gastric ulcer hemoptysis, hematochezia, skin cracking
    DaiYahejieTuberUsed for tuberculosis, tracheitis, traumatic injury, and detumescence
    De'angBageraoTuberTuberculosis, hemoptysis, bleeding from gastric ulcer, burns and scalds
    DongShaque, SanjueTuberTreat hematemesis and hemoptysis
    JingpoLahoiban, PusehzuotuberFor tuberculosis, bronchiectasis, hemoptysis, gastric ulcer, hematemesis, hematuria, hematochezia, traumatic bleeding, burns, impotence
    MengMoheeryichagan, NixingTuberFor tuberculosis hemoptysis, ulcer bleeding, traumatic bleeding, chapped hands, and feet
    MiaoBigou, Wujiu, SigouTuberUsed for hemoptysis of tuberculosis, bleeding of ulcer disease, traumatic bleeding, chapped hands, and feet
    MolaoDajiebaTuberTreat internal and external injuries caused by falls
    TibetanSanchabaijiTuberFresh chopped and soaked with honey; Powdered after sun-dried, then taken with honey and waterMainly used to treat cough, asthma, bronchitis, lung disease and a few gynecological diseases
    TuRuokeyeTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationTreatment of tuberculosis, hemoptysis, bloody stool, chapped skin
    WaBaijiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationFor tuberculosis, hemoptysis, gastrointestinal bleeding, scald and burn
    YaoBiegeidaiTuberTreat gastric ulcer, pulmonary tuberculosis, cough, hemoptysis, and hematemesis
    YiDaibaij, Tanimobbaili, Niesunuoqi, AtuluoboTuberTreatment of tuberculosis, hemoptysis, golden wound bleeding, burns, chapped hands and feet
    ZhuangManggounuTuberTreat stomachache and hemoptysis
    BaiBletilla formosana (Hayata) Schltr.Baijier, YangjiaoqiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationIt is used for emesis, hemoptysis due to tuberculosis, and hemoptysis due to gastric ulcer. External application for treatment of incised wound
    MiaoLianwuTuberThe effect is the same as that of B. striata
    LisuHaibiqiuTuberIt can treat tuberculosis, hemoptysis, epistaxis, golden sore bleeding, carbuncle and swelling poison, scald by soup fire, chapped hands and feet
    YiNiesunuoqi, Yeruomaoranruo, Atuluobo, Ribumama, Atuxixi, Abaheiji, Binyue, ZiyouTuberIt is used for tuberculosis, hemoptysis, traumatic injury, treatment of frostbite, burn, scald, bed-wetting of children and other diseases
    BaiBletilla ochracea Schltr.Baijier, YangjiaoqiTuberAfter the roots are dried, chew them orally or grind them into powder for external applicationFor hematemesis, epistaxis, hemoptysis due to tuberculosis, hemoptysis due to gastric ulcer; External application of golden sore and carbuncle
    MengMoheeryichagan, NixingtuberThe effect is the same as that of B. striata
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    The formation of traditional medical knowledge among Chinese people is often directly related to the specific living environment and cultural background[34]. For example, the Bai, Dai, De'ang, Jingpo, Lisu Yi, Wa and other ethnic minorities live in mountainous areas. The cold weather in winter and year-round outdoor manual work makes it difficult to maintain their skin[35, 36]. In the face of this situation, the ethnic people who are concerned about their physical appearance have long ago chosen local Bletilla species for skin care, and have handed down this tradition for many generations[34]. This important traditional skin care tradition is worthy of further in-depth study.

    The six main classes of Bletilla chemical components, phenanthrene derivatives, phenolic acids, bibenzyls, flavonoids, triterpenoids, and steroids, have been described previously. Almost three hundred compounds have been isolated from Bletilla, including 116 phenanthrene derivatives, 58 phenolic acids, 70 bibenzyls, 8 flavonoids, 24 triterpenoids and steroid and 13 other compounds (Figs 514). Chemical structures of the isolates of Bletilla species most are phenanthrene derivatives, which have been demonstrated to possess various pharmacological activities.

    The prominent opioids oxycodone, hydrocodone, naloxone, and naltrexone are all phenanthrene derivatives[37]. Currently, phenanthrene derivatives (Fig. 5, 1 to 66) were isolated from B. formosana, B. ochracea, and B. striata. In 2022, 17 phenanthrene derivatives (117) were isolated from the ethyl acetate (EtOAc) extracts of B. striata tubers[38]. Then, other phenanthrene derivatives were isolated from Bletilla, such as dihydrophenanthrenes (1841), phenanthrenes (4266), biphenanthrenes (Fig. 6, 6789), dihydro/phenanthrenes with uniquestructures (90112) and phenanthraquinones (Fig. 7, 113116). Thus far, this genus has been documented to include these compounds, which have been shown to exhibit pharmacological actions[3945].

    Figure 5.  Phenanthrene derivatives from Bletilla species (1−66)[3841,4345,47,49,58,7072,7479].
    Figure 6.  Phenanthrene derivatives from Bletilla species (67−105)[41,43,49,5961,70,76,7986].
    Figure 7.  Phenanthrene derivatives from Bletilla species (106−116)[43,49,70,75,84,85,87].

    Phenolic acids are carboxylic acids created from the skeletons of either benzoic or cinnamic acids[4648]. Fifty-eight phenolic acids (Figs 810, 117 to 174) were isolated from B. formosana, B. ochracea, and B. striata.

    Figure 8.  Phenolic acids from Bletilla species (117−134)[1,5,36,4752,54,67,88,90].
    Figure 9.  Phenolic acids from Bletilla species (135−169)[39,45,4854,56,61,68,69,73,76,82,83,8994].
    Figure 10.  Phenolic acids from Bletilla species (170−174)[20,95].

    For example, compounds 121, 126, 139, 141, 148, 149, 154, 155 and 157 were isolated from the rhizomes of B. formosana[1,49,5052]. The structures of these compounds were determined, mostly from their NMR spectroscopy data. Additionally, protocatechuic (136) and vanillin (137) also have been isolated from B. striata[53]. Moreover, some bioactive components such as 2-hydroxysuccinic acid (164) and palmitic acid (165) have been discovered and identified from B. striata[20,5456].

    The bibenzyls were small-molecular substances with a wide range of sources, which were steroidal ethane derivatives and resembling the structural moiety of bioactive iso-quinoline alkaloids[57].

    For example, depending on their structural characteristics, 70 bibenzyl compounds (Fig. 11, 175 to 244) can be grouped into three groups, simple bibenzyls (175186, 233238), complex bibenzyls (189225) and chiral bibenzyls (226-232, 239-244)[5860].

    Figure 11.  Bibenzyls from Bletilla species (175-244)[1,4042,47,49,50,5860,70,73,76,9699].

    Flavonoids are among the most common plant pigments. Eight bibenzyls (Fig. 12, 245 to 252) have been isolated from B. formosana, B. ochracea, and B. striata. Apigenin (245) and 8-C-p-hydoxybenzylkaempferol (249) were isolated from the whole plant of B. formosana[45]. Bletillanol A (250), bletillanol B (251) and tupichinol A (252) were isolated from B. striata[61]. The names and chemical structures of the flavonoids reported from Bletilla are shown below (Fig. 12).

    Figure 12.  Flavonoids from Bletilla species (245–252)[45,61].

    Twenty-four triterpenoids and steroids (Fig. 13, 253 to 276) have been reported from Bletilla (Fig. 13), such as, tetracyclic triterpenes (253259) and pentacyclic triterpenes (189225) and chiral bibenzyls (260)[6264]. Steroids (261276) isolated from the Bletilla and have shown some bioactivity. For example, bletilnoside A (272) was isolated from Bletilla species and displayed anti-tumor activity[65,66].

    Figure 13.  Triterpenoids and steroid compounds from Bletilla species (253-276)[56,6265].

    Thirteen other compounds (Fig. 14, 277 to 289) were isolated from B. formosana, B. ochracea, and B. striata. These compounds included amino acids, indoles and anthraquinones[67,68]. For example, syringaresinol (285) and pinoresinol (286) have been described in the methanol extract of the tubers of B. striata[61].

    Figure 14.  Others compounds from Bletilla species (277−289)[50,54,61,62,67,68,94,97].

    Based on the information about the chemical constituents of Bletilla species, it appears that there is a substantial body of research on these compounds. However, there are some areas that may warrant further investigation and research. At first, it would be valuable to investigate potential synergistic effects and interactions between the different classes of compounds within Bletilla species, as some of the compounds may work together. Besides, it is worth considering the improvement of compound yield. Optimizing extraction methods and finding the most efficient and environmentally friendly techniques are vital for both research purposes and potential commercial applications. It is also important to take into account the variability in chemical composition among different Bletilla species and even within the same species from different cultivars.

    The rich and varied chemical components make the plants of Bletilla have a wide variety of pharmacological activities (Table 4). Many studies have shown that the plants of this genus have anti-inflammatory, antineoplastic, antiviral, antioxidant, hemostatic, antibacterial, and other biological activities, which help to support the traditional medicinal practice of Bletilla in folk medicine.

    Table 4.  Summary of the pharmacological activities of Bletilla species.
    Pharmacological activityTested substance/partTested system/organ/cellTested dose/dosing methodResultsRefs.
    Anti-inflammatoryEthanol extract of Bletilla striataRAW264.7 cells RAW264.7 cells were pre-treated with ethanol extract of B. striata for 1 h and then stimulated with LPS (200 ng/mL) for 12 h, 0.05% DMSO was applied as the parallel solvent control. The culture supernatant was collected for IL-6 and TNF-α detection.Ethanol extract of B striata significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 µg/mL.[41]
    The ethyl acetate-soluble (EtOAc) extract of tubers of B. striataH2O2-induced PC12 cell injury modelPC12 cells were seeded in 96-well multiplates at a density of 1.5 × 105 cells/mL. After overnight incubation at 37 °C with 5% CO2, 10 μM test samples and H2O2 (final concentration of 450 μM) were added into the wells and incubated for another 12 h.It protected the cells with the cell viabilities of 57.86 ± 2.08%, 64.82 ± 2.84%, and
    64.11 ± 2.52%.
    [98]
    Ethanol extract of tubers of B. striataRAW264.7 cellsCells were treated with ethanol extracts (25 μM) dissolved in DMSO, in the presence of
    1 μg/mL lipopolysacchride
    (LPS) for 18 h
    The anti-inflammatory activity with IC50 of 2.86 ± 0.17 μM.[54]
    PE extract of the tubers of B. striataLPS-stimulated BV2 cellsCells treated with extract
    (0, 1, 10, 30, 100 μg/mL) and dihydropinosylvi (0, 1, 10, 30, 100 μM) in presence of LPS
    (1 μg/mL)
    The anti-inflammatory activity with IC50 values of 96.0 μM.[96]
    Ethanol extract of the roots of B. striataCox-1 and Cox-2Treated with the ethanol extracts at various concentrations
    (0, 1, 10, 100 μM)
    The compounds with sugar moieties displayed selective inhibition of Cox-2 (N90%).[38]
    B. striata polysaccharide (BSPb)Human mesangial cells (HMCs)HMCs were pre-treated with BSPb (5, 10, 20 μg/mL)BSPb efficiently mediated expression of NOX4 and TLR2, to attenuate generation of ROS and inflammatory cytokines.[12]
    Compounds extracted from the rhizomes of Bletilla ochraceaRAW264.7 cellsAfter 24 h preincubation,
    cells were treated with serial dilutions in the presence of
    1 μg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in the supernatant was assessed by adding 100 μL of Griess regents.
    It showed the inhibitory effects with IC50 values in the range of 15.29–24.02 μM.[76]
    Compounds extracted from the rhizomes of B. ochraceaMurine monocytic RAW264.7 cellsAfter 24 h preinubation, RAW 264.7 cells were treated with compounds (25 μM) dissolved in DMSO, in the prenence of
    1 μg/mL LPS for 18 h. NO production in each well was assessed by adding 100 μL of Giress regent
    It showed the inhibitory effects with IC50 2.86 ± 0.17 μM.[86]
    Compounds extracted from the rhizomes of Bletilla formosanaElastase Release AssaysNeutrophils (6 × 105 cells/mL) were equilibrated in MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM) at 37 °C for 2 min and then incubated with a test compound or an equal volume of vehicle (0.1% DMSO, negative control) for 5 min.Most of the isolated compounds were evaluated for their anti-inflammatory activities. The results showed that IC50 values for the inhibition of superoxide anion generation and elastase release ranged from 0.2 to 6.5 μM and 0.3 to 5.7 μM, respectively.[49]
    Anti-tumorTwo compounds from Bletilla striataA549 cellsCompounds were tested for their ability to induce ROS generation in A549 cells at concentrations of 20 two compounds for 24 h, the cells were harvested to evaluate the ROS production.The two compounds exhibited antiproliferative effects using the MTT test; these effects may be due to cell cycle arrest and inducing ROS generation.[87]
    Stilbenoids from B. striataBCRP-transduced K562 (K562/BCRP) cellsIt showed antimitotic activity and inhibited the polymerization of tubulin at IC50 10 μM.[78]
    Compounds extracted from the rhizomes of B. ochraceaThe human tumor cell lines HL-60 (acute leukemia), SMMC-7721 (hepatic cancer), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer)100 μL of adherent cells were seeded into each well of 96-well cell culture plates. After 12 h of incubation at 37 °C, the test compound was added. After incubated for 48 h, cells were subjected to the MTS assay.All isolated metabolites except 7 were evaluated for cytotoxic activity against five human cancer cell lines (HL-60, SMMC7721, A-549, MCF-7 and SW480).[76]
    AntiviralThe tuber of B. striataMadin-Darby canine kidney model and embryonated eggs modelAs simultaneous treatment with 50% inhibition concentration (IC50) ranging from 14.6 ± 2.4 to 43.3 ± 5.3 μM.Phenanthrenes from B. striata had strong anti-influenza viral activity in both embryonated eggs and MDCK models.[107]
    The 95% ethanol
    Extract of B. striata
    BALB/C miceIt has significant anti-influenza
    virus effect in mice, which may be related to the increase of IL-2, INFα, INF-β and thus enhance the immune function of mice.
    [12]
    AntioxidantCompounds extracted from the rhizomes of B. formosanaDPPH radical-scavenging assaySolutions containing 160 μL of various concentrations of sample extract, 160 μL of various concentrations of BHA, 160 μL of various concentrations of ascorbic acid, and the control (160 μL of 75% methanol) were mixed separately with 40 μL of 0.8 mM DPPH dissolved in 75% methanol. Each mixture was shaken vigorously and left to stand for 30 min at room temperature in the dark.Tthe seedlings grown by tissue culture of B. formosan collected in Yilan County had the best antioxidant capacity. In addition, B. formosana collected in Taitung County had the best scavenging capacity in the tubers, leaves and roots.[93]
    Fibrous roots of B. striataDPPH model and superoxide anion systemThe ABTS+ solution was prepared by reacting 7 Mm ABTS with 2.45 mM potassium persulfate (final concentrations both dissolved in phosphate buffer, 0.2 M, pH 7.4) at room temperature for 12–16 h in the dark.It removed free radicals and inhibit tyrosinase activity.[33]
    B. striata extracts (BM60)The murine macrophage cells NR8383, male SD mice (180~200 g)NR8383 were pretreated with extracts (1, 10 and 100 g/mL) for 4 h and then 65 stimulated with 1 g/mL of LPS for 24 h. Acute lung injury was induced in mice by nonhexposure intratracheal instillation of LPS (3.0 mg/kg). Administration of the BM60 extract of 35, 70, and 140 mg/kg (L, M, H) was performed by oral gavages.The BM60 treatment reduced the production of NO in NR8383 macrophages. Treatments with BM60 at the doses of 35, 70, 140 mg/kg significantly reduced macrophages and
    neutrophils in the bronchoalveolar lavage fluid (BALF).
    [12]
    The crude
    polysaccharides obtained from B. striata
    DPPH free radical scavenging activityConcentration
    range of 2.5–5.0 mg/mL
    The IC50 of BSPs-H was 6.532 mg/mL.[35]
    HemostasisB. striata polysaccharide (BSP)Diabetes mellitus (DM) mouse models were induced by high fat-diet feeding combined with low-dose streptozocin injectionDM mouse models were induced by high fat-diet feeding combined with low-dose streptozocin injection. The BSP solutions were applied on the surface of each wound at a volume of 50 μl. RD mice were assigned as normal controls and received saline treatment (n = 6). All mice were treated with vehicle or BSP once daily from the day of wounding (d0) until 12 days later (d12).BSP administration accelerated diabetic wound healing, suppressed macrophage infiltration, promoted angiogenesis, suppressed NLRP3 inflammasome activation, decreased IL-1β secretion, and improved insulin sensitivity in wound tissues in DM mice.[112]
    B. striata Micron Particles (BSMPs)Tail amputation model and healthy male Sprague-Dawley (SD) rats
    (250 ± 20 g, 7 weeks of
    age)
    Rats were divided into six groups of five treated with cotton gauze and BSMPs (350–250, 250–180, 180–125, 125–75, and < 75 μm), respectively.Compared to other BSMPs of different size ranges, BSMPs of 350–250 μm are most efficient in hemostasis. As powder sizes decrease, the degree of aggregation between particles and hemostatic BSMP effects declines.[109]
    Rhizoma Bletillae polysaccharide (RBp)Adult male SD rats weighing 220 ± 20 gAfter incubation for 1 min at 37 °C, 300 μL of PRP was dealt with different concentrations of RBp (50, 100, 150, and 200 mg/L) under continuous stirring, and the vehicle was used as the blank control.RBp significantly enhanced the platelet aggregations at concentrations of 50−200 mg/L in a concentration-dependent manner.[113]
    AntibacterialBibenzyl derivatives from the tubers of Bletilla striataS. aureus ATCC 43300, Bacillus subtilis ATCC 6051, S. aureus ATCC 6538 and Escherichia coli ATCC 11775Using a microbroth dilution method, bacteria were seeded at
    1 × 106 cells per well (200 μL) in a
    96-well plate containing Mueller-Hinton broth with different concentrations (from 1 to 420 μg/mL, 300 μg/mL and so on;
    2-fold increments) of each test compound.
    It showed inhibitory activities with MIC of (3–28 μg/mL) against S. aureus ATCC6538[116]
    The crude extract of B. striataS. album, A. capillaris, C. cassiaThey were seeded at 1 × 106 cells per well (200 μL) in a 96-well plate containing Mueller−Hinton broth (meat extracts 0.2%, acid digest of casein 1.75%, starch 0.15%) with different concentrations (from 1 to 128 μg/mL; 2-fold increments) of each test compound.It showed S. album (0.10%), A. capillaris (0.10%), and C. cassia (0.10%) to have the strongest antibacterial properties.[118]
    The ethyl acetate-soluble (EtOAc) extract of tubers of B. striataS. aureus ATCC 43300, S. aureus ATCC 6538, and Bacillus subtilis ATCC 6051) and Escherichia coli ATCC 11775)Bacteria were seeded at 1 × 106 cells per well (200 μL) in a 96-well plate containing Mueller Hinton broth with different concentrations (from 1 to 420 μg/ml; 2-fold increments) of each test compound.The extract was effective against three Gram-positive bacteria with minimum inhibitory concentrations (MICs) of 52–105 μg/ml.[98]
    The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbsS. aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 43300, E. coli ATCC 35218, and P. aeruginosa ATCC 27853, Bacillus subtilis 168EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 μg/mL.EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively.[117]
    Anti-adhesiveBletilla striata extraction solutionPPA was induced by cecal wall abrasion, and Bletilla striata was injected to observe its efect on adhesion in ratsThe rats in the sham operation group was not treated; the other rats of the three experimental groups were intraperitoneally injected with 8 ml of phosphate-buffered saline (Control group), 15% Bletilla striata extraction solution (BS group), and 0.2% hyaluronic acid solution (HA group), respectively.Bletilla striata decreased the development of abdominal adhesion in abrasion-induced model of rats and reduced the expression of the important substance which increased in PPAs.[120]
    ImmunomodulatoryB. striata polysaccharide (BSPF2)Mouse spleen cellsTo observe the immune activity of BSPF2, mouse spleen cells were stimulated with BSPF2 at 10–100 g/mL for 72 h.Immunological assay results demonstrated that BSPF2 significantly induced the spleen cell proliferation in a dose-dependent manner.[121]
    Anti-pulmonary fibrosisB. striata polysaccharideClean grade male SD ratsSD rats were randomly divided into 5 groups, sham operation group (equal volume of normal saline), model group (equal volume of normal saline), tetrandrine positive control (24 mg/kg) group and white and Polysaccharide low
    (100 mg/kg) and high (400 mg/kg) dose groups.
    The Bletilla striata polysaccharide has remarkable regulation effect on anti-oxidation system and immune system, but cannot effectively prevent lung fibrosis.[127]
    Small molecule components of Bletilla striataClean grade male SD ratsSD rats were randomly divided into 5 groups, sham operation group (0.5 mL normal saline), model group (0.5 mL normal saline), and positive control group (tetrandrine 24 mg/kg) and low (20 mg/kg) and high (40 mg/kg) dosage groups of the small molecule pharmacological components of Bletilla, which were administered by gavage once a day for 2 consecutive months.The small molecule components of Bletilla striata can effectively prevent lung fibrosis though regulating the anti-oxidation system,immune system and cytokine level; SMCBS is one of the active components of Bletilla striata on silicosis therapy[124]
    —, not given.
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    Many phytochemicals have been well characterized to lessen swelling or inflammation[89]. A series of phenolic acid and polysaccharide compounds isolated from Bletilla demonstrated anti-inflammatory bioactivity against BV-2 microglial, RAW 264.7, and PC12 cells[96,100102]. For example, phochinenin K (106) exhibited growth inhibitory effects with an IC50 of 1.9 μM, and it is a possible candidate for development as neuroinflammation inhibitory agent[43]. Using the H2O2-induced PC12 cell injury model, (7S)-bletstrin E (242), (7R)-bletstrin F (243) and (7S)-bletstrin F (244) could clearly protect the cells with the cell viabilities of 57.86% ± 2.08%, 64.82% ± 2.84%, and 64.11% ± 2.52%, respectively[98]. With an IC50 of 2.86 ± 0.17 μM, 2,7-dihydroxy-4-methoxyphenanthrene (53) showed potential action against NO generation in RAW 264.7 macrophages[54]. The use of Bletilla in traditional skin care, it is said to function as an astringent, hemostatic and wound healing[33]. Modern medical pharmacology research has validated that this plant has antibacterial effects, which may may help to explain, in part, its traditional use in skin care[24].

    Though it's mentioned that some of these compounds from Bletilla have demonstrated anti-inflammatory action, more extensive studies are needed to fully understand their mechanisms of action, potential therapeutic applications, and safety profiles. Conducting in vivo studies and clinical trials can provide more concrete evidence of their effectiveness.

    There are important antineoplastic agents that have originated from plant natural products[103]. In recent years, several bibenzyl and flavonoid compounds have been discovered from Bletilla that have antineoplastic activity against A549 cells and other cells. For example, 7-hydroxy-2-methoxy-phenanthrene-3,4-dione (160) and 3′,7′,7-trihydroxy-2,2′,4′-trimethoxy-[1,8′-biphenanthrene]-3,4-dione (163) have shown strong antiproliferative effects and induced ROS production after 24 h in A549 cells[87]. The doxorubicin (Dox)/FA (folate)-BSP-SA (stearic acid) modified Bletilla striata polysaccharide micelles boosted the drug enrichment in tumors and improved the in vivo anticancer effects[104,105]. Micelles, nanoparticles, microspheres, and microneedles are examples of B. striata polysaccharide-based drug delivery systems that exhibit both drug delivery and anti-cancer functionality. These experiments confirmed that some of the compounds isolated from the Bletilla have potential activity for the treatment of cancer.

    However, most of the evidence presented in the previous studies is based on in vitro experiments or cell culture studies. It is highly necessary to use animal models to study the in vivo anti-tumor effects of Bletilla extracts or compounds. These studies can help evaluate the safety and effectiveness of treatments based on Bletilla. Additionally, through such methods, researchers can further investigate the mechanisms of Bletilla's anti-tumor activities, exploring how Bletilla compounds interact with cancer cells, immune responses, and signaling pathways involved in tumor growth and metastasis.

    Antiviral medications are essential for preventing the spread of illness, and are especially important nowadays with pandemics and drug-resistant viral strains[5, 6]. Therefore, it is vitally necessary to find novel, safe, and effective antiviral medications to treat or prevent viral infections[106]. B. striata plant contains compounds that have been recorded in ancient texts to cure cough, pneumonia, and skin rashes, and these may be related to potential antiviral constituents[23]. Some constituents of B. striata have antiviral activity, for example, phenanthrenes and diphenanthrenes from B. striata displayed potent anti-influenza viral in a Madin-Darby canine kidney model and embryonated eggs model, diphenanthrenes with parentally higher inhibitory activity than monophenanthrenes[107]. But more research is needed to further determine the antiviral activity of Bletilla, understand how Bletilla compounds interact with viral proteins or the host immune response, and conduct safety and toxicity studies, which are crucial for the development of related materials.

    Free radicals have the potential to exacerbate lipid peroxidation and harm cell membranes, which can lead to several prevalent human diseases, including cancer, cataracts, and coronary heart disease[108]. Research has shown that extracts from Bletilla possess strong antioxidant activity. However, this antioxidant activity can vary depending on the different growing environments of the plant. Additionally, the antioxidant capabilities of extracts from different parts of the Bletilla plant also vary[93]. Clinical studies have shown that traditional Chinese medicine formulas containing Bletilla can inhibit tyrosinase activity and possess antioxidant properties, thus resulting in skin-whitening effects[108]. Furthermore, some research reveals that the polysaccharides in the plant exhibit significant antioxidant activity, effectively scavenging free radicals and inhibiting tyrosinase activity[33]. This highlights the skin-whitening potential of the fibrous root of Bletilla striata, indicating promising prospects for the comprehensive utilization of the B. striata plant[33]. However, most studies on the pharmacological activities of Bletilla have focused solely on B. striata, neglecting other species within the genus. Different species may possess varying phytochemical compositions and antioxidant properties, which can lead to an incomplete understanding of the genus as a whole.

    Available hemostatic agents are expensive or raise safety concerns, and B. striata may serve as an inexpensive, natural, and promising alternative[109]. Polysaccharides of B. striata displayed hemostatic activity through inhibition of the NLRP3 inflammasome[110112]. The ADP receptor signaling pathways of P2Y1, P2Y12, and PKC receptors may be activated as part of the hemostasis[113]. Alkaloids from Bletilla have hemostatic activities through platelet deformation, aggregation, and secretion. In addition, polysaccharides of Bletilla striata have potential wound-healing medicinal value[110]. Currently, Bletilla plants have been used in various traditional systems, such as traditional Chinese medicine and Ayurveda, to control bleeding.

    Previous studies revealed that Bletilla displayed antibacterial effects[114]. For example, bletistrin F, showed inhibitory activities with MIC of (3–28 μg/mL) against S. aureus ATCC 6538[115,116]. Antimicrobial screening of Bletilla showed S. album (0.10%), A. capillaris (0.10%), and C. cassia (0.10%) to have the strongest antibacterial properties[117,118]. In addition, phenanthrenes are worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA[119]. However, further in vivo studies on the antibacterial activity of Bletilla are lacking, which is needed for clinical application. For example, the specific mechanism of antibacterial activity of Bletilla still needs to be elucidated. While research on the antibacterial activity of Bletilla plants is promising, it faces several shortcomings and challenges that need to be addressed for a more comprehensive understanding of their potential therapeutic applications. Further studies with standardized methodologies, mechanistic insights, clinical trials, and consideration of ecological and safety concerns are essential to advance this field.

    There are other pharmacological activities of Bletilla, like anti-fibrosis activity, anti-adhesive activity, and immunomodulatory activity. For example, B. striata has been studied as a new and cheaper antiadhesive substance which decreased the development of abdominal adhesion abrasion-induced model in rats[120]. However, the natural resources of Bletilla are also getting scarcer. To preserve the sustainable development of Bletilla species, proper farming practices are required, along with the protection and economical use of these resources. The immunomodulatory activity of the Bletilla species was assessed using the 3H-thymidine incorporation method test, and BSP-2 increased the pinocytic capacity and NO generation, which improved the immunomodulatory function[121,122].

    B. striata extract was shown to have anti-pulmonary fibrosis effect[123]. B. striata polysaccharide can successfully prevent lung fibrosis through established by invasive intratracheal instillation method and evaluated by lung indexes[123,124]. Moreover, Bletilla species need further investigations to evaluate their long-term in vivo and in vitro activity before proceeding to the development of pharmaceutical formulation.

    While there is currently a deep understanding of the pharmacological activity of plants in the Bletilla genus, there are still many gaps that need to be addressed. To overcome these shortcomings, future research on the pharmacological activity of Bletilla species should emphasize comprehensive, well-designed studies with a focus on species-specific effects, mechanistic insights, and rigorous clinical trials. Additionally, collaboration among researchers, standardization of methods, and transparent reporting of results can help advance our understanding of the therapeutic potential of Bletilla plants. Researchers should also consider safety aspects and explore potential herb-drug interactions to ensure the responsible use of Bletilla-based therapies.

    There are several common clinical applications of Bletilla striata in TCM. The gum of B. striata has unique viscosity characteristics and can be used as thickener, lubricant, emulsifier and moisturizer in the petroleum, food, medicine, and cosmetics industries[125130]. B. striata is used as a coupling agent, plasma substitute, preparation adjuvant, food preservative and daily chemical raw material[131133]. In clinical practice, B. striata glue has also been proven to control the infections and is beneficial to the healing of burns and wounds[133135].

    In ethnic communities in Southwest China, the locals chew fresh Bletilla tubers directly or take them orally after soaking in honey to treat cough, pneumonia and other diseases[33, 34]. This traditional use is common in local communities in Southwest China, and suggests at the safety of Bletilla. However, current research shows it is still necessary to control the dosage when using Bletilla[136].

    Zebrafish embryos and larvae respond to most drugs in a manner similar to humans[137]. Militarine, the main active ingredient of Bletilla, was tested in a zebrafish embryo development assay at concentrations of 0.025 g/L and 0.05 g/L, and with the increased concentration, the heart rate of zebrafish embryos is slowed. Mortality and malformation rates of zebrafish embryos gradually increased with time and militarine concentration[138]. Although Bletilla species are safe at therapeutic dose ranges, further research on their safety is required[136]. More in-depth studies should be carried out on Bletilla to extract effective ingredients and make better preparations for clinical use[139].

    According to the traditional medicinal knowledge in ancient Chinese texts, Bletilla has been an important ingredient for skin care since ancient times. Many ethnic minority groups in China still retain the practice of using Bletilla for skin care, and the plant parts and preparation methods of use are consistent with the records in ancient texts. Almost 300 phytochemicals have been identified from Bletilla, and some of them possess important pharmacological activities, which support its traditional uses and suggest the important medicinal development potential of this genus. This review has demonstrated that Bletilla, as an important medicinal plant of Orchidaceae, still requires further research to fathom its medicinal potential.

    For instance, it is necessary to enhance the quality control procedures based on the chemical components and pharmacological activity of Bletilla. The chemical composition and pharmacological properties of Bletilla are critical areas of current research. According to previous studies, the main bioactive components of Bletilla can vary greatly according to its origin, harvest time, distribution, storage, and adulteration. However, variation in bioactivities caused by the differences in Bletilla constituentshave not been explored extensively yet. To develop clinical applications of Bletilla, it is crucial to further explore the mechanism of action between its chemical composition variation and its pharmacological actions.

    In addition, although the tuber has historically been the main medicinal part of Bletilla, research has shown that the chemical composition in other parts of Bletilla, such as stems, leaves, and flowers, also give these parts a variety of pharmacological activities. Further in-depth analysis of the chemical components and pharmacological activities of different parts of this genus is worthwhile, to explore the specific chemical basis of its pharmacological activities, develop related drugs, and promote clinical applications. For example, Bletilla polysaccharide has good hemostasis and astringent wound effects[110], so it may have the potential to be developed into a drug or related medical materials to stop bleeding and heal wounds.

    Finally, as a cautionary note, many unrestrained collections and the destruction of habitats have made the resources of wild Bletilla rarer. In addition to protecting the wild populations of Bletilla, appropriate breeding techniques should be adopted to meet the commercial needs of this economically important genus, thereby allowing its sustainable use in commerce.

    The authors confirm contribution to the paper as follows: study conception and design, funding acquirement: Long C; data analysis, draft manuscript preparation, literature review: Fan Y, Zhao J; manuscript revise and language editing: Wang M, Kennelly EJ, Long C. All authors reviewed the results and approved the final version of the manuscript.

    The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation, to any qualified researcher. Requests to access these datasets should be directed to Yanxiao Fan (fanyanxiao0510@163.com).

    This research was funded by the Yunnan Provincial Science and Technology Talent and Platform Plan (202305AF150121), Assessment of Edible & Medicinal Plant Diversity and Associated Traditional Knowledge in Gaoligong Mountains (GBP-2022-01), the National Natural Science Foundation of China (32370407, 31761143001 & 31870316), China Scholarship Council (202206390021), and the Minzu University of China (2020MDJC03, 2022ZDPY10 & 2023GJAQ09).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Salunke P, Metzger LE. 2023. Impact of transglutaminase treated micellar casein concentrate and milk protein concentrate on the functionality of processed cheese product slice formulations. Food Materials Research 3:31 doi: 10.48130/FMR-2023-0031
    Salunke P, Metzger LE. 2023. Impact of transglutaminase treated micellar casein concentrate and milk protein concentrate on the functionality of processed cheese product slice formulations. Food Materials Research 3:31 doi: 10.48130/FMR-2023-0031

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Impact of transglutaminase treated micellar casein concentrate and milk protein concentrate on the functionality of processed cheese product slice formulations

Food Materials Research  3 Article number: 31  (2023)  |  Cite this article

Abstract: Milk protein concentrate (MPC) is used as an ingredient in processed cheese product (PCP) formulations. However, its use can result in texture defects such as a soft body and restricted melting characteristics. The use of micellar casein concentrate (MCC), which has a higher level of casein and less serum protein, improves the texture of PCP. Further improvement in PCP products may be possible using transglutaminase (TGase), an enzyme that can crosslink proteins. This study aimed to determine the effect of TGase treatment of MPC and MCC retentates on the functionality of MPC and MCC when used in a PCP slice formulation. Three lots of MCC and MPC retentate were produced using microfiltration and ultrafiltration, respectively. Each replicate of retentate was divided into three equal portions and treated with transglutaminase enzyme at three different levels: 0.3 U/g of protein, 3.0 U/g of protein, and no TGase addition. The retentates were spray-dried, and powders were used in PCP slice formulation. Functional properties of PCP were analyzed using a penetration test, Dynamic stress rheology (DSR) for transition temperature (TT), and Schreiber melt test. As the TGase addition increased, there was a significant (p ≤ 0.05) increase in TT and a significant (p ≤ 0.05) decrease in the Schreiber melt area. The PCP made from MCC had higher TT and Schreiber melt area values than that made from MPC as an ingredient (TGase or no TGase). It was concluded that TGase treatment modifies the melt characteristics of MCC and MPC in PCP applications.

    • Processed cheese product (PCP) is manufactured in various forms such as loaves, slices, shreds, or spreads. Code of Federal Regulations (CFR) in the USA has described five major categories of processed cheese, namely pasteurized processed cheese, pasteurized processed cheese food, pasteurized processed cheese spread, pasteurized blended cheese, and processed cheese analogs, which differ in fat, moisture, pH, and ingredients that can be used[14]. PCP is an undefined category of processed cheese (PC) in the USA[1] and can further be subcategorized into substitute or imitation cheese[2]. Ingredients such as milk protein concentrate (MPC), micellar casein concentrate (MCC), and rennet casein, which are not legally allowed under CFR, are utilized in the formulation[4]. Since the PCP formulation can use various ingredients not legally allowed under the CFR, many companies prefer to tailor the functionality of PCP by using a combination of non-cheese ingredients. However, with the inclusion of non-cheese dairy ingredients, the manufacturing and chemical reactions occurring in PCP remain the same. Depending upon the type of PCP to be manufactured, the type of ingredients, emulsifying salt or protein content change, provide the specific and required functionality to the PCP. Most PCPs are manufactured using trisodium citrate (TSC), disodium phosphate (DSP), or a combination of both. TSC is primarily used in slice or slice-on-slice (SoS) applications in which the PC is cooled on a chill belt[3, 4] as it provides a desirable firmer texture, better flexibility, gloss, and reduced adhesiveness as compared to DSP. The TSC when used in PCP shows no oiling off and has better melting properties compared to other emulsifying salts[5].

      Milk proteins are essential for all structural networks and textural properties of almost all dairy products and play a critical role in their functional properties. Heterogeneous milk proteins can be fractionated into individual fractions using various membrane separation techniques. However, traditionally cheese or rennet casein powder made using chymosin has been a primary source of casein or intact casein used in PCPs. From a product and technological point of view, caseins are by far the most essential and valuable component of milk[6]. Most of the functional properties of the casein micelle depend on its surface properties[7, 8] and molecular structure[9]. Standard processing and drying techniques do not change the casein micellar structure and preserve the native properties of the micelles[8].

      Milk protein concentrate (MPC) is produced industrially using ultrafiltration. This process concentrates milk proteins (casein and serum proteins) in the same ratio as found in the milk from which it is manufactured[10]. However, lactose is removed by adding diafiltration water during processing to increase the protein content. Hence MPC has the advantage of low lactose and a high amount of soluble milk proteins. However, in certain products, such as high heat stable products, some emulsions, etc., the amount of MPC that can be utilized in a formulation is limited by the level of serum (whey) protein present in MPC. Using MPC in these formulations can result in defects, including precipitation and flocculation, resulting in undesirable product characteristics such as coagulation, poor heat stability, etc. In some applications, protein ingredients with a reduced level of serum proteins may be preferred.

      The level of serum (whey) protein in skimmed milk can be reduced using microfiltration (MF) to produce micellar casein concentrate (MCC)[1012] with altered casein-to-total nitrogen ratio. However, MPC and MCC alone cannot deliver the critical functional properties required in products such as high heat stability, emulsions, yogurt, and PCPs. The reason is that the membrane system separation involves fractionation through physical means and produces a soluble protein product; hence, the casein and serum proteins are in the micellar and native state. The casein micelles are intact, and the κ-CN, which provides steric stability to the casein micelles, still has the glyco-macro peptide (GMP) attached, providing a negative charge on its surface. This negative charge interferes with network formation in the presence of emulsifying salts and impacts product functionality[1317].

      One of the methods used to alter the functional and structural properties of milk proteins at the molecular level is by crosslinking proteins with enzymes, particularly the transglutaminase (TGase, EC 2.3.2.13) enzyme. It is used in many foods to crosslink proteins to enhance the functional properties of products. The protein crosslinking can change food's chemical, structural, textural, functional, and nutritional characteristics. Crosslinking is described as the covalent bonding of a protein to itself or other proteins in a system[1316, 18,19]. Crosslinking of proteins may help change or improve food proteins' solubility, foaming, rheological, and emulsifying characteristics[19]. The cross-linking can be intramolecular (between polypeptide chains within a protein) or intermolecular (between proteins)[1316, 20] or among CNs and whey proteins or any different proteins[1317]. The application and utilization of TGase and its basics have been reviewed[1928].

      Using a crosslinking enzyme can potentially modify the physical properties of casein, which may prevent the detrimental properties of κ-CN that are typically observed when MPC or MCC is used. TGase catalyzes the acyl transfer reaction between protein-bound glutaminyl residues and primary amines[29] and forms a covalent bond between a free amino group (i.e., lysine) and peptide-bound glutamine. It forms a framework of additional iso-peptide bonds that impact the food's functional properties, such as gelation capability, viscosity, or water binding capacity[29, 30]. Caseins have a flexible open structure, and they are an excellent substrate for TGase[31, 32] as compared to undenatured globular whey proteins[31, 33]. The high susceptibility of κ-CN towards crosslinking is likely due to its peripheral position on the CN micelles, and the high susceptibility of β-CN is due to its ease of accessibility in the micelle structure and dynamic nature[34, 35]. However, in the absence of amine substrates, TGase is capable of catalyzing the deamidation of glutamine residues[14, 24, 34, 36, 37]. The deamidation reaction can cause partial hydrolysis of proteins at high TGase levels[14, 24, 3638]. The authors used TGase in MPC and MCC to assess the changes in functionality[16] and theorized that it is possible to use transglutaminase to create a casein-based protein network from the native casein micelles in MPC or MCC that has improved functionality in various products[1317].

      Changes in functionality after TGase addition in skimmed milk or sodium caseinate solutions have been reported. Changes in heat and alcohol stability[16, 35, 3946], and emulsifying capacity[16, 34, 35, 39, 4648] have been reported in milk and sodium caseinate solutions after TGase addition. Changes in the properties of yogurt, including water holding capacity, syneresis, texture, and firmness viscosity after TGase-added milk was used for yogurt manufacture, have been reported by various researchers[16, 23, 4955]. TGase treatment has been used in fresh cheeses[56], PCP[13, 17], and imitation mozzarella cheeses[14, 15, 57].

      Conflicting reports on the impact of TGase on casein micelle structure and functionality may be related to the differing conditions (i.e. temperature, TGase concentration, micelle concentration, pH) utilized during the TGase treatments. Recently, in our laboratory PCP loaf formulation using DSP was manufactured, and changes in the functionality of PCP, especially melted characteristics, were observed[17]. Since the market for SoS is also much bigger, our experiment was planned to use TGase-treated MPC and MCC in a SoS formulation and see the impacts on melted and unmelted characteristics of PCP.

    • Three replicates of MCC and MPC were produced using MF and UF, respectively, from three different lots of milk. Each lot of retentate was divided into three equal portions, and each one was subjected to TGase treatment which included: control (C, no TGase), low (L, 0.3 U/g protein), and high (H, 3.0 U/g protein). TGase levels were selected to cover wide cross-linking possibilities and how this will impact the functionality of PCP, compared to our previous study[13], which used seven TGase units per gram of protein. Each treated retentate was spray-dried separately to obtain powders. These individual powder samples obtained were used as ingredients in a PCP SoS formulation manufactured in a rapid visco analyzer (RVA). A 2 x 3 factorial design experiment with two product types (MPC and MCC) and three TGase enzyme levels, namely control (C), low (L), and high (H), were used. Details of the experimental design are given in Fig. 1.

      Figure 1. 

      Experimental design for the manufacture of treatment powders. Processed cheese product (slice) treatment: MCC-C = micellar casein concentrate powder, control; MCC-L = micellar casein concentrate powder, low TGase level; MCC-H = micellar casein concentrate powder, high TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level; MPC-H = milk protein concentrate powder, high TGase level.

    • Raw milk received was cold separated (6 °C) at the SDSU Dairy Pilot plant, and the skimmed milk obtained was batch pasteurized (63 °C for 30 min) and cooled to 4 °C. The pasteurized skimmed milk (SM) was divided into two equal lots. Each SM lot was used for pilot-scale production of MCC and MPC using spiral wound membranes in an MF/UF unit with two single long housings arranged in parallel. Three replicates of MCC and MPC were produced using three different lots of milk. MF (0.5 µ polyvinylede fluoride) and UF (10 kD polyether sulfone spiral wound membranes) were procured from Dominick Hunter Filtration Divison – N.A (Parker Hannifin Corporation, Oxnard, CA, USA). The MF and UF operations were performed at 23.3 °C with a final retentate volume of 45.4 L resulting in a volume reduction ratio (VRR) of approximately 5 (on a feed volume basis). The MF was performed at a TMP of 86.2 kPa with DF water added at six different intervals totaling 100% (on a feed volume basis) to control the retentate viscosity and maximize serum protein removal. The UF was performed at 276 kPa TMP with DF water added at four different intervals totaling 40% (on a feed volume basis) to control the retentate viscosity and remove soluble solutes. Details of the process are discussed in our previous paper[10].

      After dividing each lot of MCC and MPC retentate into three equal portions, they were treated with TGase, (Activa TI, Ajinomoto Food Ingredients LLC, Chicago, IL, USA, activity of 100 units/g). For each TGase treatment, the required quantity TGase enzyme was weighed and mixed with 100 mL of distilled water, with details of the procedure given in our previous paper[14]. After enzyme addition to the retentate and thorough mixing, each treatment was incubated at 50 °C for 25 min. The retentates were heated to 72 °C for 10 min to inactivate the enzyme and then cooled to 4 °C. All retentates were then spray dried (ASO 412E, Niro Inc., Columbia, MD, USA) with inlet air temperature maintained at 205 °C and outlet temperature maintained at 90 °C. Powders were collected in plastic bags (Associated Bag Company, Milwaukee, WI, USA) and stored at room temperature until further analysis was completed.

    • The MPC and MCC powders were used as ingredients in SoS PCP formulations. The PCP formulations for all the replicates were developed and balanced using Techwizard (an Excel-based formulation software program by Owl Software, Columbia, MO, USA) to have 20.0% total fat, 48.0% moisture, 1.26% salt, and 17.5% protein with the treatment powders utilized contributing 15.0% protein to the formulation. The other ingredients included extra sharp premium natural aged cheddar cheese (Cabot Creamery Cooperative, Cabot, VT, USA), enzyme-modified cheese (Bongards' Creameries, Bongard, MN, USA), salted butter (Great Value, Wal-Mart Stores, Inc, Bentonville, AR, USA), trisodium citrate (KIC Chemical Inc., New Paltz, NY, USA), dibasic sodium phosphate (Rhodia Inc., Cranbury, NJ, USA), sorbic acid (KIC chemical Inc, New Peltz, NY, USA), deproteinized whey powder (Agropur Inc., Le Sueur, MN, USA), lactic acid 85% w/w (Fisher Scientific, Fair Lawn, NJ, USA), and iodized salt (Great Value, Wal-Mart Stores, Inc, Bentonville, AR, USA). The cheddar cheese had a composition of fat (31.5%), moisture (42.0%), lactose (0.8%), salt (1.8%), and total protein (25.01% with intact CN 16.47% and 4.6 pH soluble N 8.54%). The final selected detailed ingredient blend formulations are shown in Table 1.

      Table 1.  Ingredient blend formulations utilized to manufacture the six Process cheese product (slice) treatment.1

      IngredientMCC-CMCC-LMCC-HMPC-CMPC-LMPC-H
      % (Wt./Wt.)
      Water40.6340.5540.6240.5040.4540.72
      Salt1.251.251.251.251.251.25
      Tri sodium citrate2.002.002.002.002.002.00
      Whey deproteinized6.987.076.866.026.085.75
      Lactic acid1.001.001.001.001.001.00
      Butter (salted)21.4921.4821.5521.6621.6421.65
      Sodium phosphate, Dibasic0.500.500.500.500.500.50
      EMC paste0.750.750.750.750.750.75
      Sorbic acid0.200.200.200.200.200.20
      Cheddar cheese5.005.005.005.005.005.00
      Treatment powder20.2120.2020.2721.1321.1221.18
      Total100.00100.00100.00100.00100.00100.00
      1 Processed cheese product (slice) treatment: MCC-C = micellar casein concentrate powder, control; MCC-L = micellar casein concentrate powder, low TGase level; MCC-H = micellar casein concentrate powder, high TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level; MPC-H = milk protein concentrate powder, high TGase level.
    • Each formulation was used to prepare a pre-blend (200 g) by weighing and mixing all the ingredients (except disodium phosphate) in a mixer (Kitchen Aid, St. Joseph, MI, USA) for 30 min (Table 1). Each formulation was then manufactured in a Rapid Visco Analyzer (RVA-4, Newport Scientific, New South Wales, Australia)[13, 17]. The required amount of disodium phosphate was weighed in each RVA canister (Perten Instruments Inc, NA, Springfield, IL, USA) along with 15 g of the pre-blend and 0.5 g of water. The blend of ingredients was mixed at 1,000 rpm at 95 °C for 2 min and then mixed at 160 rpm for 1 min. Twelve batches of PCP were produced from each pre-blend, and the continuous viscosity profile obtained from each batch was collected. The apparent viscosity at the end of manufacture (VAM) was measured as the average of the last five points on the viscosity profile. The 12 batches were divided into various subsamples for analysis (four for penetration hardness, three for DSR, and the remainder for other analysis). For the penetration test, molten PCP immediately after manufacture in RVA was poured into a plastic X-plate (100 × 15 mm, Fisherbrand, Fisher Scientific, Pittsburgh, PA, USA) and cooled at 4 °C until further analysis was completed[17]. For DSR analysis and the modified Schreiber melt test, the PCP samples were poured into plastic molds (30 mm diameter). They were cooled to room temperature and then transferred to 4 °C until further analysis was completed.

    • The pH of each sample was measured in duplicate with a pH meter (Corning pH meter 340, Corning Incorporated, NY, USA) with an Accumet® - gel-filled glass electrode with a spear tip (Fisher Scientific, NJ, USA). The proximate analysis of the PCP samples, including moisture, fat, and total protein, was carried out using standard methods[58].

    • The penetration hardness test was performed using a TA.XT2 Texture Analyzer (Texture Technologies Corp., Scarsdale, NY, USA; Stable Microsystems, Godalming, UK) as described by Salunke et al.[17]. In this test, the sample (15 g and 10 mm thick) of PCP in an X-plate was placed directly under the probe, and each quarter of the plastic X-plate served as a replicate. The samples were tempered to 15 °C, and a uniaxial penetration was performed with a TA-8, 6.35 mm diameter stainless steel ball, and a penetration depth of 3 mm with a crosshead speed of 0.8 mm/s.

    • For DSR analysis, the PCP samples were removed from the plastic mold, and the DSR test was carried out[15]. PCP samples were prepared by cutting a thin slice (2.0 mm) using a food slicer (Model 1042W, The Rival Co. Kansas City, MO, USA) and wire cutter. Cylindrical cheese samples 28.3 mm in diameter were cut with a cork borer. The cheese samples were tempered to 20 °C prior to DSR analysis. DSR analysis was performed using a rheometer (Viscoanlyser, ATS Rheosystems, Rheologica Instruments Inc., NJ, USA) with parallel plate geometry of 30 mm diameter. The maximum stress limit for the linear viscoelastic region was obtained as 400 Pa from the stress sweep experiment. Subsequently, a temperature sweep was performed using the same rheometer (parallel plate geometry) at 1.5 Hz frequency and constant stress of 400 Pa (linear viscoelastic region). The exposed cheese surface was coated with vegetable oil (Crisco pure vegetable oil, The J M Smucker Co., OH, USA) to minimize drying during the temperature sweep. A temperature ramp from 20 to 90 °C at a 1 °C/min rate was completed to measure the rheological properties. Each sample's measurements were taken in triplicate and included the elastic modulus (G′) and viscous modulus (G″). The transition temperature (TT) and tangent angle (tan δ) were calculated. The TT was defined as the lowest temperature during the temperature gradient experiment, where tan δ equaled 1 (G′ = G″).

    • The meltability of each PCP sample was measured using a modified Schreiber test[15]. Each PCP sample was cut into a 28.5 mm diameter and 7 mm height cylinder and tempered to 20 °C for 10 min prior to analyses. Four samples from each treatment were placed on four 0.95 mm thick aluminum plates (100 mm × 100 mm) and immediately transferred to a forced draft oven (Fisher Scientific) at 90 °C. After 7 min, the plates with the melted cheese were cooled to room temperature. The diameter of the melted cheese was measured using a Vernier caliper at four locations and the average recorded. Meltability of the PCP was reported as the change in the area of the melted cheese in millimeters squared relative to the original area.

    • Collected data was analyzed using the Proc GLM factorial analysis of SAS (SAS Institute Inc., Cary, NC, USA) with a Type I error rate (α) of 0.05 to test for significant differences among treatments. This study utilized a 2 × 3 factorial design with two products (MCC or MPC) and three TGase enzyme levels (control, low and high). In addition, the product x enzyme level interaction was also tested. Mean value comparisons were made at a 0.05 level of significance using least significance difference (LSD), and results were considered significant at p < 0.05.

    • Mean PCP composition, including moisture, fat, protein, and pH of the six PCP treatments, are shown in Table 2. Minor differences were observed in moisture, fat, protein, and pH, however, the differences were not statistically significant (p > 0.05). These results were expected because all the replicates used in this study were balanced for moisture, fat, salt, protein, and lactose using various ingredients mentioned in Table 1.

      Table 2.  Mean (n = 3) composition of Processed cheese product (slice) treatments.1

      ParametersMCC-CMCC-LMCC-HMPC-CMPC-LMPC-H
      pH5.705.645.695.705.745.66
      Fat (%)19.9319.9419.9719.9919.9519.97
      Protein (%)17.5217.4917.4917.4917.5117.55
      Moisture (%)47.9547.9647.9447.9647.9547.98
      1 Processed cheese product (slice) treatment: MCC-C = micellar casein concentrate powder, control; MCC-L = micellar casein concentrate powder, low TGase level; MCC-H = micellar casein concentrate powder, high TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level; MPC-H = milk protein concentrate powder, high TGase level.
    • The functional properties of PCP were analyzed using an RVA for viscosity after manufacture (VAM), penetration test for penetration hardness, dynamic stress rheology (DSR) for transition temperature (TT), and Schreiber melt test for change in melt area. Mean squares and P values (in parentheses) of VAM, penetration hardness, TT, and melt area of the six PCP manufactured using MPC and MCC are shown in Table 3. There was a significant replicate effect in TPA-hardness (p < 0.05), which may have resulted from variations in manufacturing conditions among the replicates. There was a significant (p < 0.05) effect of product type and enzyme level in all PCP functional properties (VAM, melt area, TPA-hardness, and DSR-melt temperature) (Table 3). There was also a significant (p < 0.05) interaction effect of product type x enzyme level in all PCP functional properties except penetration hardness (Table 3). The product type and enzyme level significance (p < 0.05) indicates a change in all PCP functional properties. In contrast, the significance (p < 0.05) of the interaction term product type x enzyme level indicates that these changes were not linear.

      Table 3.  Mean squares and p values (in parentheses) of RVA viscosity, melt area, hardness, and tan delta of the PCP manufactured from treatment powders.

      FactorsDfRVA viscosityChange in areaHardnessTan delta
      Replication2771,444.57 (0.085)56,935.00 (0.089)5,511.35 (0.002)*16.71 (0.328)
      Product type1308,265.16 (0.005)*405,215.28 (0.0008)*22,552.99 (<0.0001)*317.24 (<0.0001)*
      Enzyme level214,197,912.72 (<0.0001)*565,898.42 (<0.0001)*2,740.38 (0.021)*417.53 (<0.0001)*
      Product type x Enzyme level2139,446.25 (0.022)*262,920.46 (0.001)*775.23 (0.239)142.79 (0.003)*
      Error1024,246.6118,276.87466.9713.37
      * Statistically significant at p < 0.05.
    • The apparent viscosity is continuously measured during PCP manufacture in the RVA, and the VAM can be used as a measure of cheese viscosity during manufacture. The mean values of the VAM of the six PCP are indicated in Table 4. PCP manufactured using MPC-C or MPC-L had significantly (p < 0.05) higher VAM than the MCC-C and MCC-L treatments. In both the MCC and MPC treatments, there was no significant (p < 0.05) difference in manufacturing viscosity between PCP made using the low level of TGase and control (MCC-C vs. MCC-L and MPC-C vs. MPC-L). PCP manufactured with high TGase levels (MCC-H or MPC-H) had a significantly higher (p < 0.05) VAM as compared to their respective control and low TGase treatments. However, there was no significant difference (p < 0.05) between the MPC-H and MCC-H treatments. Both products (MCC and MPC) and enzyme levels (C, L, or H) affected VAM.

      Table 4.  Mean (n = 3) RVA viscosity, melt area, hardness, and tan delta of the PCP manufactured from treatment powders at 3 levels (C, L, and H).

      ParameterMCC-CMCC-LMCC-HMPC-CMPC-LMPC-H
      RVA viscosity (cP, n = 36)728.7c ± 47.2725.5c ± 72.53653.1a ± 143.31204.3b ± 269.81122.4b ± 144.73565.7a ± 276.9
      Change in area (mm, n = 12)1081.9a ± 203.4708.4b ± 29.361.3e ± 26.7412.0c ± 44.8323.4cd ± 104.4215.8d ± 241.0
      Hardness (g, n = 12)402.1b ± 33.4456.5a ± 54.3398.0b ± 50.4340.7c ± 35.1359.8c ± 21.9343.8c ± 14.7
      Tan delta (°C, n = 9)65.97c ± 3.7769.60b ± 3.7890.00a ± 0.0061.97d ± 2.9467.99bc ± 4.8870.42b ± 4.75
      a–e Means in a row with common superscripts do not differ (p ≥ 0.05). Processed cheese product (slice) treatment: MCC-C = micellar casein concentrate powder, control; MCC-L = micellar casein concentrate powder, low TGase level; MCC-H = micellar casein concentrate powder, high TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level; MPC-H = milk protein concentrate powder, high TGase level.
    • The penetration hardness measures unmelted cheese firmness using a texture analyzer. The mean values of penetration hardness of the six PCP are shown in Table 4. The PCP manufactured using MCC had a significantly higher (p < 0.05) hardness than the respective MPC treatment. As the level of TGase enzyme increased, there was an increase in penetration hardness. However, PCP made from high TGase levels (MCC-H and MPC-H) showed lower penetration hardness values even though they seemed to be hard visually and physically. This may be due to the formation of tiny spherical particles and possibly because it had higher interstitial air pockets causing the penetration probe to penetrate at a lower force. In addition, MCC-H and MPC-H had higher levels of crosslinking, and the MCC-H sample also had hydrolyzed peptides[14,16]. Both products (MCC and MPC) and enzyme levels (C, L, or H) affected the firmness of the PCP.

    • The modified Schreiber melt test measures the melt and flow (spread) behavior of PCP. The mean values of melt area (change in the area) of the six PCP are shown in Table 4. PCP manufactured using MCC-C or MCC-L had a significantly (p < 0.05) higher melt area than that of MPC-C and MPC-L. MCC-C had the highest melt area and was significantly (p < 0.05) higher than the other samples. The addition of TGase (MCC-L) significantly (p < 0.05) reduced melt area, but it still had a higher melt area than MCC-H and all MPC samples. PCP having higher meltability will have more melt area and vice-versa. At the higher TGase level, MCC-H showed very little melt and was significantly (p < 0.05) lower than MPC-H. However, melt area decreased with TGase treatment in both MPC and MCC.

    • The rheological characteristics of PCP samples, G', G", G*, and viscosity obtained by dynamic stress rheology are shown in Figs 2 & 3. The MCC-C sample had higher G' and G" values as compared to the MPC-C sample (Fig. 2), with similar results observed for the G* and viscosity results (Fig. 3). The MCC-C being higher in casein showed lower G', G", G*, and viscosity values after 80 °C whereas MPC-C having more serum proteins melted at 70 °C. The samples with higher TGase (MCC-H and MPC-H) showed higher G', G", G*, and viscosity than other samples, indicating TGase crosslinking affected the viscoelastic properties of cheese. The samples with lower TGase (MCC-L and MPC-L) had rheological characteristics between the control and higher TGase samples.

      Figure 2. 

      Rheological characteristics of PCP (G' and G") of process cheese samples. Processed cheese product (slice) treatment: MCC-C = micellar casein concentrate powder, control; MCC-L = micellar casein concentrate powder, low TGase level; MCC-H = micellar casein concentrate powder, high TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level; MPC-H = milk protein concentrate powder, high TGase level.

      Figure 3. 

      Rheological characteristics of PCP (G* and viscosity) of process cheese samples. Process cheese product (slice) treatment: MCC-C = micellar casein concentrate powder, control; MCC-L = micellar casein concentrate powder, low TGase level; MCC-H = micellar casein concentrate powder, high TGase level; MPC-C = milk protein concentrate powder, control; MPC-L = milk protein concentrate powder, low TGase level; MPC-H = milk protein concentrate powder, high TGase level.

    • The mean values of DSR-melt temperature (TT) of the six PCP are shown in Table 4. The PCPs manufactured with MCC had higher TT values than the corresponding MPC. The MPC-H, MPC-L, and MCC-L had similar TT values. The PCP made using control (MCC-C and MPC-C) showed the lowest TT values. The TT of the MPC-L was between the MCC-C and MCC-L. Similar to the melt area data, the MCC-H significantly differed from the other treatments. The PCP made using MCC-H did not melt at 90 °C as it was crosslinked too much. Both products (MCC and MPC), as well as enzyme levels (C, L, or H), affected the melt area and TT.

    • After TGase treatment and drying, MCC and MPC were used as ingredients for manufacturing PCP using the SoS formulation (Table 1). Differences in the product (MPC and MCC) and TGase level had a significant effect on the unmelted (hardness) and melted (VAM, melt area, rheology, and TT) characteristics of PCP.

      The MCC and MPC formulations were balanced for total protein content (Tables 1 & 2); hence the MCC provided a higher level of intact CN and less serum protein, whereas MPC had a lower level of intact CN and a higher level of serum protein. The difference in the constituents was because of inherent processing differences between MF and UF to produce MCC and MPC, respectively[10]. Compared to MPC, the MCC retentate was significantly (p < 0.05) higher in TN/TS ratio[10]. In addition, MCC powder protein and calcium contents were higher than the MPC powder, whereas the lactose content was almost 50% less than that of MPC powder[14].

      Both product type and enzyme level affected the VAM (Tables 3 & 4). The PCP manufactured using MPC had a higher viscosity than those manufactured using MCC (Table 4). The PCP VAM was affected by the level of intact casein and serum proteins. The VAM results from various protein interactions with protein and fat[59]. These interactions are dependent on the characteristics of the protein that forms the structure of the PCP[60], and the presence of intact (unhydrolyzed) CN which results in a fibrous CN network[3,4,13,17,61,62]. The additional serum protein in the MPC treatments can aggregate due to the high cook temperature (95 °C) used during PCP manufacture. This aggregate can cause an increase in viscosity[4,13,17,62]. Similar observations were noted by other researchers and concluded that the denaturation of serum or whey protein during PCP manufacture forms a heat-induced irreversible gel and produces a PCP with restricted melt characteristics[3,4,61,62]. Even though the MPC had higher VAM than the MCC samples, the hardness and meltability were lower.

      Post-manufacturing melt characteristics were measured by a modified Schreiber melt test, rheology, and TT, whereas unmelted characteristics were measured using a penetration test. The cheese melt is defined as cheese's ability to flow and spread[15, 63], and a modified Schreiber melt test can be used to measure the meltability. Additionally, DSR was used to measure the rheological parameters (G', G", G*, and viscosity) given in Figs 2 & 3. The TT was calculated where tan δ (G″/G′) = 1 and is a convenient measure of the melting point of PCP because this is the lowest temperature where a material changes from primarily elastic to primarily viscous[4] and has been used to quantify the melting characteristic of PCP[15]. A cheese that melts easily and quickly will have a low TT and vice-versa[15].

      The extensive crosslinking of SP and casein at higher temperatures[4, 64], during PCP manufacture in formulations having MPC causes restricted melt characteristics. The meltability[4,15,62,65] and firmness[4,14,62,65] of PC foods decreased significantly with an increased amount of WP and were attributed to the emulsification and denaturation of SP[4,62,65]. However, others found similar results but concluded that the SP inhibits meltability but is not the only cause of the melt defect[66]. As SP concentration increased, the fibrous structures became apparent, responsible for the loss of cheese meltability[66].

      Penetration hardness is a direct indication of the firmness of PCP. Even though the VAM was higher for PCP containing MPC, its penetration hardness values were less, indicating a soft body. The use of high intact CN cheese in PCP formulation has been shown to increase the hardness of PCP[3,4,14,62]. The higher hardness in samples having MCC in the formulation has been reported, and the differences were attributed to higher intact CN and lower serum protein content in MCC used[14,62]. The use of MPC resulted in a soft body and restricted melting characteristics (Table 4). It was because of the difference between intact casein and serum protein in MPC and MCC powders used in the PCP formulation.

      The MCC and MPC retentates were treated with TGase before drying[14,15], which significantly changed the functionality of the SoS PCP. The TGase action caused a high amount of inter-and intra-protein crosslinking in MCC and MPC samples, contributing to these changes[1316]. The treatments with TGase addition at high levels (MCC-H and MPC-H) crosslinked protein fractions via covalent bonding[14,16]. As a result, they changed the surface properties of CN micelle, causing changes in viscosity, emulsification capacity, and water holding capacity[14,16], which in turn caused an increase in VAM. We theorize that the strong covalent crosslinking at the high TGase level in MCC-H and MPC-H caused a significant increase in VAM. This covalent interaction is difficult to break and may have caused different protein-protein interactions and network formation, affecting VAM. Authors have reported changes in VAM after TGase treatment in PCP[13] and loaf-type PCP[17]. Other researchers have also reported increased viscosity after TGase treatment in other product matrices, such as yogurt, because of TGase crosslinking[51,52,55].

      Similarly, higher covalent crosslinking due to TGase action modifies CN and SP at the molecular level causing restricted melt characteristics[13,15,17]. The casein molecules (their number, strength, and type of bond) are fundamental building blocks of rheological properties[15,63], and the melting of cheese is primarily determined by the number and strength of CN-CN interactions above 50 °C[15,67]. The covalent bond is the most robust bond linkage, which is very difficult to destroy in the conditions encountered during PCP manufacture. The MCC-H and MPC-H had higher levels of crosslinking[1316] and low meltability, while MCC-L and MPC-L had a low level of crosslinking and hence had more meltability as measured by melt tests.

      The PCP made using MCC-H did not melt at 90 °C as it was crosslinked too much[14,15]. These results are supported by the Schreiber melt test (Table 4) and rheology (Figs 2 & 3), as described above. As expected, samples with a low melt area had a high TT, and samples with a high melt area had a low TT. The CN-CN interactions were so strong in MCC-H that the matrix showed no signs of melting, even at the highest temperature. MCC, primarily intact CN, could be the reason for high TT in PCP. At elevated temperatures (> 40 °C), the CN-CN interaction (the number and strength) determines melting behavior[63,67]. Hydrophobic interactions, electrostatic repulsion, hydrogen bonds, CCP crosslinks, and disulfide bonds all play a critical role in cheese melt characteristics[63]; the TGase covalent bonding role needs further investigation. A further detailed study is required at a molecular level to determine the effect of such covalent bonds.

      Overall, the use of MCC and MPC in SoS formulation affects viscosity during manufacture, texture, and melt characteristics. TGase treatment changes the functionality of MCC and MPC in SoS PCP formulation with an increase in viscosity during manufacture at higher levels of TGase usage and also restricts melt.

    • The unmelted and melted functional properties of PCP were affected by TGase treatment. The MPC samples had higher viscosity during manufacture as compared to MCC samples. The MPC samples showed restricted melt and softer texture compared to MCC samples. As the TGase addition increased, there was a significant (p ≤ 0.05) increase in TT and a significant (p ≤ 0.05) decrease in the Schreiber melt area. Rheological studies also confirmed the results. The PCP made from MCC had higher TT and Schreiber melt area values than that made from MPC as an ingredient (TGase or no TGase). It was concluded that TGase treatment modifies the melt characteristics of MCC and MPC in PCP slice formulations. By controlling the crosslinking new PCP products can be manufactured that meet specific functional requirements.

      • We thank Dairy Research Institute (Rosemont, IL, USA) and Midwest Dairy Foods Research Center (St. Paul, MN, USA) for funding this project.

      • The authors declare that they have no conflict of interest.

      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press on behalf of Nanjing Agricultural University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (3)  Table (4) References (67)
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    Salunke P, Metzger LE. 2023. Impact of transglutaminase treated micellar casein concentrate and milk protein concentrate on the functionality of processed cheese product slice formulations. Food Materials Research 3:31 doi: 10.48130/FMR-2023-0031
    Salunke P, Metzger LE. 2023. Impact of transglutaminase treated micellar casein concentrate and milk protein concentrate on the functionality of processed cheese product slice formulations. Food Materials Research 3:31 doi: 10.48130/FMR-2023-0031

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