Investigating the characteristics of killer yeasts and their applications in food: a review
Subject Areas :Azita Faraki 1 , MohammadReza Rezaeifard 2
1 - Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
2 - Department of Food Industry and Technology, Faculty of Veterinary Medicine, University of Semnan, Semnan, Iran
Keywords: Killer yeasts, Biocontrol agents, saccharomyces cerevisiae, protein or glycoprotein toxins, bacteriocin activity,
Abstract :
Yeasts have been discovered and used for many years. They are so valuable and practical. In addition to their benefits in food production, their use as a biocontrol and protective agent is considered a great advantage. Among the yeasts in the environment, there is a group called killer yeasts, which, according to research, play an important role in controlling other microorganisms (other yeasts or bacteria). Killer yeasts can be classified in different aspects, but in general, the most important known killer yeast is saccharomyces cerevisiae. The toxins secreted by these yeasts have the role of inhibiting the target organisms in the environment. The nature of these toxins is protein or glycoprotein. Each toxin has its mechanism, and sometimes they are very complicated; there are two important strategies for killer yeasts: (1) competition for food in the environment and (2) penetrating the target cell membrane and destroying it. The most important applications of killer yeasts are fermentation, fundamental biological research, biomedical research, food and feed ingredients, and biocontrol agent. Many studies have been conducted on killer yeasts in various fields, such as the destruction and inhibition of microorganisms in winemaking, bacteriocin activity against pathogens, and antimicrobial activity in food spoilage, fruits, and vegetables. In vivo studies have also been conducted. All these cases indicate the killing activity of killer yeasts and their use as a suitable alternative. It should be mentioned that the dose of killer yeast is so essential. Also, their excessive presence causes problems and safety issues must be considered. Finally, it should be noted that the applications of killer yeasts in the food industry and other fields are increasing and only future studies will demonstrate their real potential of them.
Abstract
Yeasts have been discovered and used for many years. They are so valuable and practical. In addition to their benefits in food production, their use as a biocontrol and protective agent is considered a great advantage. Among the yeasts in the environment, there is a group called killer yeasts, which, according to research, play an important role in controlling other microorganisms (other yeasts or bacteria). Killer yeasts can be classified in different aspects, but in general, the most important known killer yeast is saccharomyces cerevisiae. The toxins secreted by these yeasts have the role of inhibiting the target organisms in the environment. The nature of these toxins is protein or glycoprotein. Each toxin has its mechanism, and sometimes they are very complicated; there are two important strategies for killer yeasts: (I) competition for food in the environment and (II) penetrating the target cell membrane and destroying it. The most important applications of killer yeasts are fermentation, fundamental biological research, biomedical research, food and feed ingredients, and biocontrol agent. Many studies have been conducted on killer yeasts in various fields, such as the destruction and inhibition of microorganisms in winemaking, bacteriocin activity against pathogens, and antimicrobial activity in food spoilage, fruits, and vegetables. In vivo studies have also been conducted. All these cases indicate the killing activity of killer yeasts and their use as a suitable alternative. It should be mentioned that the dose of killer yeast is so essential. Also, their excessive presence causes problems and safety issues must be considered. Finally, it should be noted that the applications of killer yeasts in the food industry and other fields are increasing and only future studies will demonstrate their real potential of them.
Keywords: Killer yeasts, Biocontrol agents, saccharomyces cerevisiae, protein or glycoprotein toxins, bacteriocin activity.
Introduction
As you know, humans have discovered and used yeasts for thousands of years. These microorganisms are so essential and beneficial for the environment. They are fungi that grow as single cells. They reproduce by budding, and their classification is as follows: Ascomycetes (e.g., Pichia, Candida, and Saccharomyces) and Basidiomycetes (e.g., Trichosporon, Rhodotorula, and Cryptococcus). The taxonomy and distinguishment of yeasts are affected by several factors such as (I) the size and shape of the cell, (II) the mechanism of budding and the formation of daughter cells, (III) the structure of hyphae (IV) the attendance of sexual spores, and (V) the presence or absence of capsules (1,2). Based on a taxonomic study by Kurtzman et al, the number of recognized yeast species is almost 1500 (3).
Among yeasts, there is a group called killer yeasts. These germs were first discovered in 1963 by Bevan and Makower. They understood the killer activity of specific species of Saccharomyces cerevisiae (against sensitive strains of the same species) (4). These organisms can secrete different types of exotoxins in terms of structure and function. The nature of these toxins is protein and glycoprotein (5,6,7). What’s more, killer toxins have differences that include the following: molecular size, mature structure, structural genes, and immunity (8). The process of producing killer toxins seems to be an intra-species or inter-species strategy that makes an environment for microbial competition. This feature has been reported in more than 100 species (belonging to more than 20 genera), and the number of cases identified through research is regularly increasing (9). They are naturally found in fruits and vegetables. As a result, the presence of these organisms has a significant effect on the other flora (10). Further research showed that killer yeasts and their toxins use resources and kill other susceptible cells (the same or other susceptible species). In fact, the target cells of killer yeasts are not only fungi but also bacteria or protozoa (11).
In this paper, we will review the features and applications of killer yeasts. Furthermore, this study aims to gather the results of the most important studies in this field.
Classification and most important killer yeasts
The classification of killer yeasts can be based on the structure of their toxins. The structure of killer toxins includes three categories: (I) small single-subunit proteins, (II) dimeric (hetero) proteins, and (III) multimeric protein complexes. Moreover, there is another classification based on gene localization and the genetic basis of toxin production (12). Besides, Scientists isolated different types of killer phenotypes from various genera of yeasts (13). The most important species of killer yeasts are Saccharomyces cerevisiae, Kluyveromyces lactis, Hanseniaspora uvarum, Debaryomyces hansenii, Pichia kluyveri, Candida krusei, and Zygosaccharomyces bailii. According to studies conducted by scientists, Saccharomyces cerevisiae has the best toxin system (14). Another important killer yeast is Kluyveromyces lactis. The killer system of this specie is related to linear DNA plasmids. The number of subunits is three, which are matured in the Golgi complex (15).
The mechanisms of killer yeast toxins
Each toxin has its mechanism to identify and kill sensitive cells. As we mentioned before, toxins of killer yeasts are protein or glycoprotein compounds that can affect the same or closely related species. The function of these toxins is similar to the activities of bacteriocin in bacterial species (16). The main feature of killer yeasts is antagonistic interactions and creating a competitive environment. Most of the time, their mechanism of action is penetration into the cell membrane. They can bind to special receptors on the cell surface and then enter into it (12). The conspicuous point is that these yeasts have intrinsic immunity. In other words, their toxins do not kill themselves (17). Some studies reported that the responsibility for producing some toxins belongs to double-stranded DNA viruses located in plasmids, cytoplasm, and chromosomal genes of yeast cells (18). In recombinant DNA technology, killer plasmids of S. cerevisiae and K. lactis can be useful as cloning vectors for the secretion of polypeptides (19). The killer system mechanism of Kluyveromyces lactis seems to be the inhibition of adenylate cyclase in target cells. With this action, the target cells lose their viability (20). Next, the mechanism of one of the most well-known killer yeasts, Saccharomyces cerevisiae, is reviewed.
1. RNA Virus
The toxin of this specie is encoded by double-stranded RNA viruses and other killer systems like KHR1 and KHS1 genes (21,22). L-A virus is an influential virus and encodes the toxin by dsRNA. This virus uses the chromosomal genes of yeast for survival and spreads among cells throughout the budding of yeasts, but it does not release into the environment (23,24). Types of dsRNAs (M1, M2, and M28) encoded killer toxins (K1, K2, and K28) of Saccharomyces cerevisiae. These toxins are inherited by cytoplasm and encapsidated in virus-like particles (25).
2. Toxin
As we previously mentioned, the dsRNA of viruses in yeast cells plays an essential role. This dsRNA translated and produced an initial protein, which is called preprotoxin. To produce the α/β dimer (an active form of the toxin), the preprotoxin is processed and cleaved, then released into the environment (17,26).
The function of killer yeast toxins
The function of killer yeast toxins is summarized in Figure 1. These toxins are divided into four groups according to their action on the target cell. (I) Toxins can create membrane pores, for example, the toxins of T1-type (K1, K2, and PMKT). They use β-1,6-glucan as the principal cell wall receptor. (II) Toxins of T2-type demonstrate glucanase activity and cause cell destruction and lysis. (III) Toxins cause problems in the cell nucleus. (IV) Toxins that target the cellular RNAs. T3 and T4 toxins have intracellular targets. They are placed in groups III and IV, respectively (27).
Applications of killer yeasts
Nowadays, scientists have paid much attention to the applications of yeasts as bio-protective agents in different fields such as agriculture, medical science, the food industry, and biotechnology (like fermentation). Also, they are suitable as model systems for studying their mechanisms (28). Because of the antagonistic activity of killer yeasts and the death of sensitive cells by them, many in vivo and in vitro studies have been conducted by scientists (29). As we mentioned, yeasts have a significant role in the food industry and biotechnology; for example, providing different types of products, including the production of alcoholic beverages, beer, wine, bread, and fermented food (30). The most notable applications of yeasts in biotechnology are presented in Figure 2 (31,32).
Killer yeasts can be beneficial in different situations, especially those diseases caused by fungi. Also, they are employed for food production. Nowadays, the indiscriminate use of antibiotics has increased the resistance of various microorganisms. Therefore, one of the best and most important strategies is to use natural resources such as yeasts and killer yeasts. This explanation refers to the word bio-preservation. Killer yeasts demonstrated significant antagonistic activity against other organisms and showed this feature during the period of production and preservation (33,34). Additionally, they can reduce the contamination of the fermentation process and can be used as a starter culture (35). On the other hand, in some cases, killer yeasts and their toxins have caused problems in the industry and commercial processes. They can kill useful and desirable strains. Therefore, all of them are not beneficial (36). The summary of characteristics of the most important killer toxins (for S. cerevisiae and K. lactis) is presented in Table 1 (28).
Results and Discussion
Lots of research about killer yeasts have done by scientists. The results of their study are very worthwhile and remarkable. Next, we will review the results of the most important articles in different fields.
1. Winemaking
Some species are autochthonous, which means that they have the potential to control food spoilage without any interference. In a study, the killing activity of autochthonous yeasts was investigated by Ullivarri et al. They confirmed that using killer yeasts as a starter culture in winemaking would allow producing high-quality wines (37). In a study by Mehlomakulu et al, the aim was to isolate novel killer toxins secreted by wine-related non-Saccharomyces yeasts. These toxins can act as bio-preservatives against Brettanomyces spp. The results of this study presented that two new killer toxins, CpKT1 and CpKT2 of two different strains of Candida Pyralidae, have the potential to control Brettanomyces strains such as B. bruxellensis. Notably, these toxins are not affected by environmental parameters such as low pH ranges, temperatures below 25 °C, sugar, and ethanol concentrations. Their research revealed that these killer toxins do not affect the bacteria responsible for wine production and also can control the growth of the microbial population in grape juice or wine (38). Due to the widespread use of killer yeasts and their toxins in the food industry and agriculture, Carboni et al produced a lyophilized toxin of killer yeast in winemaking. This killer yeast toxin was named Kpkt and produced by Tetrapisispora phaffii. After preparing the desired species and applying it to grape must, a significant killer activity was observed on wild wine-related yeasts. Also, this toxin showed a powerful and remarkable antimicrobial activity against lactic acid bacteria and foodborne pathogens. Based on these findings, bioreactor production and lyophilization can be novel and beneficial methods in the winemaking and food industries (39). In another research related to winemaking, the purified killer toxin of Saccharomyces eubayanus (SeKT) decreased the content of volatile phenols (the production of spoilage yeasts such as Brettanomyces bruxellensis, Pichia membranifaciens, Meyerozyma guilliermondii, and Pichia manshurica). The possible mechanism of this toxin against sensitive strains is through the activity of β-glucanase and chitinase enzymes. This action caused damage to the cell wall and the death of sensitive species. It is worth noting that the death resulting from cell necrosis and apoptosis depends on the toxin dose. So, they reported that SeKT is a suitable Candidate to control spoilage yeasts in winemaking (40). Villalba et al isolated a new killer toxin produced by Torulaspora delbrueckii. They examined 18 wine strains of T. delbrueckii and among them, the strain T. delbrueckii NPCC 1033 named TdKT showed a positive effect on the yeasts that cause spoilage in wine. Moreover, glucanase and chitinase enzymatic activities were observed. TdKT increased necrosis and apoptotic cell death after 3 and 24 hours, respectively. It showed that the mechanism of this action is dependent on time. The findings of their study demonstrated that killer toxin extracts are active in oenological conditions. They proved the ability of this toxin to control the spoilage of products such as wine (41). To control Brettanomyces bruxellensis in winemaking, Santos et al, use Ustilago maydis killer toxin as a new agent for managing wine spoilage. They isolated 39 strains of Saccharomyces cerevisiae and Brettanomyces bruxellensis from wineries and tested them against 39 killer yeasts. They observed the killing activity of U. maydis for the first time. The results showed that U. maydis CYC 1410 could inhibit B. bruxellensis, whereas this strain does not affect S. cerevisiae. This toxin was active at pH ranges between 3 and 4.5 and temperatures between 15 °C and 25 °C, proving the ability to control decay during winemaking. In addition, a small amount of killer toxin extract can decrease 4-ethylphenol and volatile phenols that produced by B. bruxellensis (42).
2. The role of Saccharomyces cerevisiae toxins
K1 and K28 are the most valuable toxins of S. cerevisiae. There is a receptor on the cell wall of the target, which is named β-1,6-D-glucan. K1 binds to the receptor, passes through the cell membrane, and then binds to the plasma membrane receptor (Kre1p). The presence of this toxin shapes a cation-selective ion channel in the membrane that is lethal for the target cell (43). On the other hand, K28 enters the target cell by the α-1,6-mannoprotein receptor. This toxin moves into the cell and stops DNA synthesis. Therefore, it causes apoptosis (44,45). The results of a study claimed that the K1 toxin inhibits the TOK1 membrane potassium channel in the cell wall. As a result, this action deactivates TOK1 as a receptor (46). On the other hand, Breinig et al, reported that the TOK1 channel is not the primary receptor for the K1 toxin. So, inhibition of the TOK1 channel does not provide immunity to killer toxins (47). Vališ et al, examined the cells that produce K1 toxin but are not immune to it. The results showed that their toxins will destroy the cell membrane receptors. This action can be due to the lack of α chain processing (48). Although Saccharomyces cerevisiae has shown significant killing activity, the presence of some species of this yeast causes spoilage in food. Therefore, the control of these species can be done by other killer yeasts. In research by Liu and Tsao, the effect of Williopsis Saturnus var. Saturnus (a known killer toxin-producing yeast) was studied as a bio-preservative specie against spoilage yeasts in cheeses. This killer yeast inhibited the growth of galactose-fermenting yeast (S. cerevisiae VL1) inoculated at ∼103 CFU/g and inhibited the growth of lactose-fermenting and galactose-fermenting yeast Kluvyveromyces marxianus (inoculated at ∼103–104 CFU/g). On the other hand, these two spoilage yeasts grew to ∼106 CFU/g from the initial cell count of ∼103 CFU/g without the killer yeast. The results indicated that W. Saturnus var. Saturnus can be an efficient specie as a food preservative (49).
3. Antimicrobial activity against food, beverage, and fruit spoilage
As previously mentioned, another killer yeast is the Williopsis Saturnus. In a research, the interactions among 17 killer yeast strains and pathogenic fungi were investigated. The results of their study indicated that the simultaneous usage of several types of killer yeasts could have a positive and significant effect on the control of fungal pathogens (50). Labbani et al, reported that a novel killer protein (Pkkp) of Pichia kluyveri yeast has antimicrobial activity against food and beverage spoilage yeasts. This strain was isolated from Algerian soil. Also, the MIC of purified Pkkp showed high antimicrobial activity against Dekkera bruxellensis and Saccharomyces cerevisiae. According to the results of this study, this killer protein can be used as a new food-grade compound to control food spoilage (51). The killer effect of yeasts and their compounds on other microorganisms has shown different results. For example, in a study by Palpacelli et al in 1991 killer strains of the genera Saccharomyces, Hansenula, and Kluyveromyces were investigated against harmful yeasts in the food industry. The results showed that Saccharomyces strains killed only Zygosaccharomyces rouxii strains, while non-Saccharomyces strains have a wider anti-yeast activity (52). In a study on Malaysian fermented food, 29 yeasts showed killing activity against some Candida species. These killer yeasts include 22 strains of Trichosporon asahii, four isolates of Pichia anomala, and one isolate of Pichia norvegensis, Pichia fermentans, and Issatchenkia orientalis, respectively. They demonstrated that the presence of these organisms in fermented food creates a competitive circumstance. These killer yeast strains produce toxins or other toxin compounds in competition for limited nutrients and space (53). The effect of killer yeasts on molds is so considerable. One of the most important causes of postharvest decay in orange fruit is Penicillium digitatum. This specie was tested against two killer yeasts such as Saccharomyces cerevisiae and Wickerhamomyces anomalus. The killing activity of W. anomalus is due to the presence of β–glucanase. Actually, this enzyme causes damage to the hyphal of P. digitatum. The results of this study demonstrated that the disease severity decreased to 1 and 4%, respectively, during the storage time (10 days). The usage of this novel method in which fruits such as oranges are purposely inoculated with pathogens and protected from decay by killer yeasts is a considerable improvement in the field of food preservation (54). In 2010, Dubash et al isolated five species with killing activity among eight yeasts. These species include Saccharomyces cerevisiae, Candida pintolopesii, Candida tropicalis, Pichia anomala, and Dekkera spp. They observed a significant amount of killer toxin activity against sensitive strains. They suggested that finding a killer toxin from a non-pathogenic species can be very useful in overcoming fungal diseases. Also, this killing activity can considerably reduce the cost of food storage (55). As already indicated, the killing activity of some yeast strains can control the decay in fruits. In a study by Lima et al, 580 yeast strains isolated from tropical fruits, 29 species had the killer phenotype. The ability of these strains was tested to control Colletotrichum gloeosporioides germination in vitro assay. The results demonstrated that five yeast strains could significantly reduce the mycelial growth and conidial germination of C. gloeosporioides. Among these five cases, Meyerozyma guilliermondii was the most effective strain, which could reduce the fungal mycelial growth (60%) and block conidia germination (100%). Their research indicated that the deployment of killer yeasts from tropical fruits could control decay caused by C. gloeosporioides in Papaya. They also suggested that bio-control techniques can be so helpful for managing the disease in tropical fruits (56). In a study by Perez et al, the killing activity of native yeasts was investigated in lemon fruit (as biocontrol agents of postharvest fungal diseases). They stated that the emergence of resistant strains due to the utilization of synthetic fungicides causes irreparable and dangerous effects, so they tried to use native yeasts to control decay in lemon fruit. According to their report, among 437 yeast strains, 8.5% showed a killer phenotype. The most identified species belong to the genera Saccharomyces, Wickerhamomyces, Kazachstania, Pichia, Candida, and Clavispora. These killer yeasts were tested against pathogenic strains such as Penicillium digitatum and P. italicum in vitro and in vivo assays. The results of this study demonstrated that two strains of Pichia and one strain of Wickerhamomyces could control the decay in lemon fruit, significantly (p<0.05). Therefore, these native killer yeasts can be a worthy alternative for the biocontrol of postharvest fungal diseases (57). In a study conducted on Botrytis cinerea strains, 42 yeast strains of 20 different species were analyzed in vitro assay for antimicrobial activity against 18 pathogenic B. cinerea strains. The most important inhibitory species were Pichia membranifaciens, P. anomala, and Debaryomyces hansenii. Also, P. membranifaciens CYC 1106 inhibited apple wounds. They reported that certain and special killer yeasts or their toxins might have the potential as novel biocontrol agents (58).
4. Antimicrobial activity against pathogens
As already indicated, some yeast species can produce antimicrobial compounds. So, they can inhibit some of the pathogenic and spoilage bacteria. In 2017, some yeast species including Candida kefyr, Saccharomyces cerevisiae, Candida intermedia, Candida tropicalis, Candida lusitaniae, and Candida krusei isolated and identified from milk and meat products. The antimicrobial activity of these species against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli was investigated. The results showed high activity with C. intermedia against S. aureus and E. coli, C. kefyr against E. coli, and C. lusitaniae against S. aureus. Moderate activities were observed with C. tropicalis, C. lusitaniae, and S. cerevisiae against E. coli. It is worth noting that all species in this study revealed an extremely low antimicrobial activity against P. aeruginosa (59). Using alginate biofilms containing killer yeasts is one of the best and most important research in this field. This new method was investigated by Blaszczyk et al on organic apples. Organic fruits are so sensitive to fungal decay because they are produced without synthetic fungicides. According to their proposal, alginate biofilms containing Pichia membranifaciens and Wickerhamomyces anomalus (as killer yeasts to control Botrytis cinerea and Penicillium italicum species) seem to be an appropriate solution for the control of postharvest pathogens (60). It is clear that the killer yeasts cannot overcome all microbial species. In a study by Meneghin et al, the antibacterial activity of killer yeast strains against those bacteria contaminating the alcoholic fermentation process (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum, and Leuconostoc mesenteroides), was investigated. Their findings showed that the target bacteria were not inhibited by any Saccharomyces cerevisiae killer strains (5 out of 11). They suggested that the killer activity of yeasts might control the population of contaminating bacteria as biocontrol agents. The real antibacterial activity against gram-positive bacteria must be confirmed by a larger group of S. cerevisiae (61).
5. Harmful effects
Although killer yeasts have positive characteristics, it should be emphasized that their excessive presence causes problems. Two types of Saccharomyces killer yeast (K2 and Klus) were found by Maqueda et al, in spontaneous wine fermentation from southwestern Spain. They reported that special attention should be taken in commercial winemaking to prevent the harmful effects of the killer phenotype because the amount of killer yeast increased significantly compared to sensitive yeast. These actions caused significant problems during the fermentation progress (62).
6. The effect of other compounds on killer yeasts activity
As we mentioned earlier, killer yeasts have a significant impact on the microbial flora of the environment or food. But it is worth noting that this function can be more effective in the presence of other compounds. Halotolerant killer yeasts were isolated from fermented foods (such as miso, soy sauce, and salted vegetables) by Suzuki et al. They investigated the characteristics of this group of killer yeasts and concluded that killer yeasts change their activity in the presence of NaCl. These strains are widely spread in Japanese foods (including salts) and laboratory samples (63). In another study, the effect of salt on the killer phenotype of yeasts from olive brine was investigated by Llorente et al. Their results showed that salt (0, 3, or 6%) can increase the killing action of killer yeasts against sensitive cells. In addition to increasing the sensitivity of the target cells, there was no significant effect on the amount of toxin production. They suggested that salt can be a suitable additive for killer yeasts in competition with sensitive cells (64).
7. In vivo models
In addition to in vitro studies, research has been conducted in vivo models similar to human intestinal conditions. Among 22 kinds of cheese, 42 isolates of Debaryomyces hansenii were isolated and evaluated for killing activity against Candida albicans and Candida tropicalis (under different ranges of pH and temperature). Among these strains, 23 cases showed killing activity against target bacteria. Also, a cell-free mycocin demonstrated killing activity against C. albicans at 35 °C and pH 6.5 (physiological conditions in the human gastrointestinal tract). Their observations indicated that D. hansenii could affect Candida populations in the gut (65).
Conclusion
According to the results of studies, using killer yeasts in the agriculture, medicine, and food industry is almost a novel method. Due to the applications of the secreted toxin of killer yeasts (with a protein or glycoprotein nature), they can inhibit the sensitive strains of other yeasts or bacteria (10). Different phenotypes of killer yeasts have complex killing mechanisms, so more studies are needed to investigate them (66). The use of killer yeasts in preventing spoilage of fruits and vegetables is known as an effective protection method during both pre and postharvest stages. Also, their effect on pathogens is so significant. Among killer yeast strains, Saccharomyces cerevisiae is very important and practical, especially in winemaking. It is worth noting that sometimes the presence of killer yeasts can be harmful, so it is necessary to control them. Although many studies have been done in this field and have shown the advantages of killer yeasts, commercial formulations and exploitation on the industrial level are still not available, therefore more studies are needed. Also, more in vivo studies should be done (67,68). Actually, only future studies will demonstrate the real potential of killer yeasts in different fields. This important knowledge increases the use of killer yeasts as biocontrol agents against a wide range of microorganisms (69,70). Finally, it should be noted that the applications of killer yeasts in the food industry and other fields are increasing, and the market demand is an excellent motivation to continue and increase research and development in this field.
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