March 30, 2021 Longchang Chemical

Earlier we mentioned the conversion of rutin and isoquercitrin. Among the various conversion methods, the conversion method with the best yield and purity is the use of enzyme-catalyzed methods, such as α-L-rhamnosidase by microorganisms and Hesperidinase. And α-L-rhamnosidase is usually combined with β-D-glucosidase to form naringinase to play a catalysis role, and in early studies, researchers equated the concept of “α-L-rhamnosidase” with “Ningrinase”. So let’s first introduce the basic knowledge of naringinase.

Naringinase can hydrolyze bitter substances such as naringin and hesperidin in citrus fruits, so it is used for debittering citrus juices, and is named after it. The main bitter component in citrus fruits is naringin, which can be degraded by naringinase in two steps: in the first step, α-L-rhamnosidase hydrolyzes naringin into rhamnose and purunin; in the second step, β-D-glucosidase further hydrolyzes prunin into naringenin and glucose without bitter taste. The hydrolysis mechanism is shown in Figure 1. Among them, purunin contains only one-third of the bitterness.

Figure 1 The hydrolysis mechanism of naringin by naringinase

As early as 1938 and 1958, naringinase was obtained from celery seeds and grapefruit leaves by Hall and Ting, respectively. After that, researchers obtained naringinase from other animals and plants. In addition to animals and plants, naringinase is more widely present in microorganisms. The naringinase currently used in research and industrial production is also mainly derived from microorganisms. Among them, natural fungi are the main source of naringinase, such as Aspergillus niger, Aspergillus oryzae and Penicillium. A small amount of naringinase is derived from yeast. And some other naringinase are derived from bacteria, which enzymatic properties and application scope have a big difference to those from fungi. Table 1 shows the naringinase and its properties of different sources studied by some scholars.

Table 1 Naringinase from different sources and their properties

SourcesStrainsSubstrateOptimum temperature /°COptimumpHMolecular weight /kDa
plantCelery seedsNaringin
Grapefruit leavesNaringin504.0
Fagopyrum esculentump-NPR、Rutin70
animalTurbo cornutusNaringin、p-NPR、Rutin2.8, 4.5~5.0
Pig liverdiosgenin427.047
bacteriaSphingomonas sp. R1Naringin508.0110
Thermomicrobium sp.p-NPR707.9, 5.0~6.9104, 107
Pediococcus acidilacticip-NPR、Rutin、hesperidin50,705.5, 4.574, 241
Brevundimonas sp.Naringin20~376.0~7.0
Bifidobacterium dentiump-NPR、Naringin、Rutin、Poncirin、ginsenoside356.0100
fungusAspergillus nigerNaringin、Rutin、hesperidin40~604.0~5.065
A. kawachiiNaringin、p-NPR、hesperidin504.090
A. oryzaeNaringin、p-NPR、hesperidin、neohesperidin455.023
Penicillium decumbensNaringin、p-NPR、Rutin7.0120
P. corylopholumNaringin、Rutin576.567
yeastPichia angustaNaringin、Rutin、hesperidin、quercitrin406.090
Cryptococcus laurentiiNaringin
Williopsis californicaNaringin

Comparing the properties of naringinase derived from bacteria and fungi in Table 1, it can be seen that although the molecular weight of naringinase derived from fungi is lower than that of bacteria, it is more suitable for reaction under acidic conditions, so it is suitable for the debittering of citrus juice; and for those naringinases derived from bacteria, the optimum pH environment for glycosidase is moderate or weakly alkaline, and it has a wider reaction temperature and good temperature stability.

With the continuous deepening of research and the discovery of naringinase with different properties, the enzyme has been widely used in medicine, food and cosmetics. The initial application was the debittering of citrus juices. Naringin is the main bitter substance in citrus juices. Its bitterness threshold in water and juice is about 20 ppm, and its threshold can reach 50 ppm in some citrus juices. It shows that when its content reaches 1.5 ppm, it will make people feel bitter. Therefore, in the juice processing of citrus and other fruits, debittering treatment is an indispensable process. Naringinase is a high efficiency enzyme that can hydrolyze naringin and other bitter substances, and naringinase can achieve the purpose of debittering well. Huang Gaoling et al. used naringinase to debitter the Guanxi honey pomelo juice and hydrolyze it at 60 ℃ and pH 3.6 for 100 min. The debittering rate of the juice can reach more than 97%. Chen Hong et al. used Aspergillus aculeatus JMUdb058 to obtain naringinase by solid state fermentation, and used it for the debittering of fruit juices. The debittering rate was as high as 99.6%, and a very good debittering effect was obtained.

At the same time, because naringinase contains α-L-rhamnosidase, it can be used to specifically produce rhamnose and purunin. Rhamnose is a kind of methyl pentose. It can be used as a drug intermediate to synthesize cardiotonic and spice Furaneol. It can also synthesize flavors and at the same time can be used as a sweetener. It can also be used as an intestinal penetration test agent. It has obvious anti-cancer effect. Wei Shenghua et al. used naringinase and yeast resting cells as catalysts to transform naringin by a two-step biological method to prepare rhamnose crystals with a mass fraction greater than 98.5%. Purunin as a kind of flavonoids, have unique functions in the field of immune, anti-cancer, anti-viral and antioxidant activities. Therefore in the field of food and medicine industries, purunin has important application value. Hu Qunfang and others used solid-state fermentation to produce α-L-rhamnosidase, and carried out biotransformation of naringin under suitable conditions, and the content of Pruning in the product was more than 95%.

Also using the reactive activity of naringinase, naringinase can further be used to improve the flavor of wine. In the alcohol brewing process, a variety of microorganisms will produce some free volatile substances and non-volatile precursors. α-L-rhamnosidase first decomposes these non-volatile precursors to obtain monoterpenoid β-D-Glucoside, then β-D-glucosidase continues to decompose to release monoterpenoids, which have a significant effect on enhancing the flavor of wine. Manzanares et al. used the rhamnosidase gene rha A encoded by Aspergillus aculeatus to be cloned and expressed in yeast, and used together with β-D-glucosidase produced by other strains for the fermentation of wine, resulting in a significant increase in aroma substances in wine. Specific data as shown in picture 2.

Figure 2 Application of naringinase in wine fermentation

In addition, naringinase can also be used to produce antibiotics and convert flavonoids. For example, Chloropolysporin is a deglycosylated glycopeptide antibiotic that has a strong inhibitory response to Gram-positive bacteria. Sankyo et al. found that the activity of rhamnosidase in naringinase can be used to synthesize the antibiotic, and found that the combined use of chloropolysporin C antibiotics and lactam antibiotics can effectively enhance its antibacterial effect on Staphylococcus. Beekwilder et al. obtained rhamnosidase from a lactic acid bacterium Lactobacillus plantarum and used the enzyme in the fermentation of tomato pulp. They found that it can remove the rhamnose in tomato pulp and enhance the biotransformation reaction of flavonoids. Therefore, the lactic acid bacteria may increase the biotransformation rate of flavonoids in the human digestive system. Hu Fuliang et al. found that propolis flavone glycosides can be degraded by naringinase to synthesize aglycones, thereby enhancing its antioxidant activity.

In summary, naringinase has very broad application prospects. In order to increase the reusability and stability of naringinase and reduce industrial production costs, naringinase is generally fixed on a carrier before the reaction. In the next article, we will focus on the enzyme fixation method for your reference.

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