Comparative Study on Functional Properties of Mulberry Black Tea Fermented by Phellinus igniarius and Eurotium cristatum

Yanrui MA, Xuezhen LI, Xuemei YANG, Guangpeng LIU, Le CHU, Yan ZHAO, Fatao HE*

1. Jinan Fruit Research Institute, All China Federation of Supply & Marketing Co-operatives, Jinan 250200, China; 2. Shandong Agricultural University, Taian 271000, China

Abstract [Objectives] To compare functional properties of mulberry black tea fermented by Phellinus igniarius and Eurotium cristatum. [Methods] The functional components of mulberry black tea fermented by P. igniarius and E. cristatum, the deep processing and health care effects of two probiotic fungi were explored. [Results] The polysaccharide content in the tea fermented by P. igniarius was the highest at 0.410 mg/mL, and the hydroxyl radical scavenging rate was the strongest, reaching 60.31%; the content of flavonoids in the tea fermented by E. cristatum was the highest, reaching 60.65 μg/mL, and the content of polysaccharides was 83.17% higher than that of mulberry black tea, reaching 0.279 mg/mL. [Conclusions] The mulberry black tea fermented by P. igniarius and E. cristatum has improved in different functional components, and has its own advantages in aroma and mouthfeel, so it has certain research value and market prospect.

Key words Phellinus igniarius, Eurotium cristatum, Mulberry black tea, Functional components

1 Introduction

Phellinusigniariusbelongs toPhellinusin Hymenochaetaetaceae of Hymenochaetales, Agaricomycetes in Basidiomycota. It is a precious medicinal fungus, the fruiting body is mostly yellow-brown, and it is a hard large fungus that grows on a variety of trees. According to the different parasitic tree species, the color, shape and composition ofP.igniariusare also slightly different. At present, there are 15 species ofPhellinusspecies known in the world, and 10 species in China[1], among which the most well-known ones areInonotushispidusandSanghuangprousvaninii.P.igniariusis first recorded inTreatiseonPropertiesofMedicinals. It has the functions of promoting blood circulation, hemostasis, resolving fluid retention, and checking diarrhea. With the development of modern medicine, more pharmacological effects ofP.igniariushave been gradually discovered. The latest research shows thatP.igniariushas obvious anti-cancer and anti-tumor effects, can effectively inhibit the growth and metastasis of tumor cells, and will not cause any toxic and side effects to the human body[2-8]. At present,P.igniariusis mostly eaten by people in the form of fruiting bodies, the eating method is single and the mouthfeel is poor. Therefore, it is urgent to develop a deep-processed product ofP.igniariusso that it can be accepted by more consumer groups.

Eurotiumcristatumbelongs to Eurotium in Trichocomaceae, Eurotiales, Eurotiomycetes in Ascomycota. It is a dominant strain in Chinese traditional Fu Tea Brick. It can give Fu Tea Brick a special aroma, and has the functions of reducing lipid and glucose, improving the function of human digestive tract, and has antibacterial and antioxidation effects[9-10]. With the development of microbiology and fermentation engineering, people have become more and more aware of the broad application value ofE.cristatumin improving the quality of fermented tea, improving the application level of food biotechnology, especially in intestinal microecological regulators, it has broad application prospects[11].

As a traditional Chinese drink, tea has a wide range of consumer groups. At present, the processing technology of Fu Tea Brick withE.cristatumas the dominant strain in China is relatively mature, but there are few reports on the research onP.igniarius. Therefore, in this experiment, we intended to use mulberry black tea as the culture substrate, separately inoculatedP.igniariusandE.cristatum, and compared and studied the change rules of different functional components.

2 Materials and methods

2.1 Materials and reagents

2.1.1Sources of the strains. The fermentedPhellinusigniariusstrain D was provided by Shandong Linqing Zheshuo Agricultural Products Co., Ltd., andEurotiumcristatumstrain was screened and purified from commercially available Anhua dark tea.

2.1.2Culture medium and reagents. PDA culture medium, purchased from Qingdao Binde Biotechnology Co., Ltd.; PDB medium, purchased from Beijing Solarbio Science & Technology Co., Ltd.; 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from TCI (Shanghai) Chemical Trading Co., Ltd.; Anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.; sodium carbonate was purchased from Tianjin Hengxing Chemical Reagent Manufacturing Co., Ltd.; Folin-Phenol was purchased from Shanghai Macklin Biochemical Co., Ltd.; sodium nitrite and aluminum nitrate were purchased from Tianjin Damao Chemical Reagent Factory; sodium hydroxide was purchased from Sinopharm Chemical Reagent Co., Ltd.

Mulberry black tea culture medium: mulberry black tea was provided by Shandong Linqing Zheshuo Agricultural Products Co., Ltd.; after rehydration, 15 g of mulberry black tea was weighed and placed in a petri dish, and sterilized by high pressure steam to prepare the mulberry black tea culture medium.

2.2 Instruments and equipmentBSC-150 constant temperature incubator and YXQ-LX-100A high pressure steam sterilizer were products of Shanghai Boxun Industry & Commerce Co., Ltd.; SW-CJ-2FD clean bench was products of Suzhou Antai Air Technology Co., Ltd.; UV1000 ultraviolet Spectrophotometer was provided by Shanghai Techcomp Instrument Ltd.; HH-4 constant temperature water bath was products of Jintan Jieruier Electric Co., Ltd.

2.3 Experimental methods

2.3.1Activation of strains and preparation of seed solution. Took thePhellinusslant strains preserved in the laboratory, used an inoculation shovel to take about 0.5 cm2in the clean bench and inoculated it to the PDA solid medium, and placed it in a 28 ℃ incubator for constant temperature cultivation for 7-10 d. After the strain covered the entire plate, stored in a refrigerator at 4 ℃ for later use. Took the above activated strain plate, used a hole puncher to take the same size of bacteria block in the clean bench and inoculated it into the sterilized PDB medium, and placed it in a constant temperature shaking incubator at 28 ℃, and cultured at 180 r/min for 72 h. The activation ofE.cristatumand the preparation of seed solution are the same as those ofP.igniarius.

2.3.2Preparation of fermented mulberry black tea. Took the above-mentioned activatedP.igniariusandE.cristatumstrain plate, used a puncher to take the same size of the bacteria block in the clean bench and inoculated it in the sterilized mulberry black tea medium, and placed it at 28 ℃ for cultivation. Incubated at a constant temperature for 7-10 d until the bacteria grew on the tea surface. The fermented mulberry black tea was dried overnight in a 45 ℃ incubator.

2.3.3Determination of hydroxyl radical scavenging rate. We determined the scavenging rate of hydroxyl radicals using the method of Xia Fenghuaetal.[12]with a slight improvement, ground the dried fermented black tea into powder, and added 60% ethanol to the sample powder at a ratio of 30∶1, conducted ultrasonic extraction at 30 ℃ for 30 min, placed in a centrifuge at 3 500 r/min for 10 min, transferred the supernatant to a 50 mL centrifuge tube, added 60% ethanol to the precipitate again. Conducted the extraction again under the same conditions, and combined the two extracts to obtain the ethanol extract of fermented mulberry black tea.

Took 8 mmol/L of FeSO4solution and 1 mL of salicylic acid solution and mixed well. Took 0.1 mL of the ethanol extract prepared in the previous step, added it to the mixed solution and shook well. Then, added 1 mL of 3% H2O2solution, shook well to make it fully react. Placed the mixture in a water bath and kept at 37 ℃ for 30 min, and then measured the absorbance at the wavelength of 510 nm. The formula for calculating the hydroxyl radical scavenging rate (P, %) is:

P=[1-(A1-A2)/A0]×100%

(1)

whereA1denotes the absorbance value of the sample reaction system,A2denotes the absorbance value of deionized water instead of H2O2reaction system, andA0is the absorbance value of deionized water instead of ethanol extraction reaction system.

2.3.4Determination of DPPH free radical scavenging rate. We determined the DPPH free radical scavenging rate using the method of Jin Qunlietal.[13]with a slight improvement. Precisely weighed 5 mg of DPPH reagent, dissolved it in 250 mL of absolute ethanol, and prepared the DPPH solution with a concentration of 0.02 mg/mL. Took 0.1 mL of the ethanol extract prepared in the previous step and mixed it with 3.9 mL of DPPH solution evenly, protected from light for 20 min at room temperature, and then measured the absorbance at the wavelength of 517 nm. The formula for calculating the DPPH free radical scavenging rate (Z, %) is:

Z=[1-(A1-A2)/A0]×100%

(2)

whereA1denotes the absorbance value of the sample reaction system,A2denotes the absorbance value of the reaction of anhydrous ethanol instead of DPPH solution, andA0is the absorbance value of the reaction system of anhydrous ethanol instead of alcohol extraction.

2.3.5Determination of polyphenol content. Took 1 mL of ethanol extract and put it in a test tube, added 1 mL of Folin-Phenol reagent, mixed well by vortex, and placed at room temperature for 3 min; then added 1 mL of 10% Na2CO3solution, mixed well by vortex, placed in a constant temperature water bath at 30 ℃ for 30 min, and mixed well by vortex; finally measured the absorbance value at the wavelength of 760 nm.

2.3.6Determination of flavonoid content. Took 1 mL of ethanol extract and put it in a 10 mL graduated test tube, added 0.3 mL of sodium nitrite solution with a mass concentration of 5%, and then placed it for 6 min; added 0.3 mL of aluminum nitrate solution with a mass concentration of 10%, and placed for 6 min; added 4 mL of sodium hydroxide solution with a concentration of 1 mol/L, diluted to the mark with 60% ethanol, placed in the dark for 15 min, and then measured the absorbance at the wavelength of 510 nm.

2.3.7Determination of polysaccharide content. Took 1 g of fermented tea sample, put it in a 50 mL centrifuge tube, added 25 mL of 80% ethanol, ultrasonic extracted for 30 min, centrifuged at 4 000 r/min for 10 min, and discarded the supernatant. Added 40 mL of distilled water to the precipitate, extracted in a boiling water bath for 2 h, then performed suction filtration, washed the residue 2 to 3 times with water, and diluted the filtrate to 100 mL for later use. Diluted the sample by 2 times, pipetted 1 mL into the test tube, added 1 mL of 5% phenol, quickly added 5 mL of concentrated sulfuric acid, placed for 10 min, then oscillated by vortex, reacted in a water bath at 30 ℃ for 20 min, and then measured the absorbance at the wavelength of 490 nm.

3 Results and analysis

3.1 Analysis of functional components and antioxidant activityPolysaccharide is the main biological active substance ofP.igniarius, and it has biological activities such as anti-tumor, hypoglycemic, liver protection, immune regulation, and antioxidant[14]. Tea is rich in polyphenols, with more than 30 kinds of components, mainly including flavonoids, glycosides, flavanols, flavonols,etc., and most of them have pharmacological effects. In addition, tea polyphenols have strong antioxidant activity and can produce obvious synergistic effects with antioxidants such as VEand VCin tea[15]. Therefore, we used polysaccharide, polyphenol, flavonoid content, DPPH· scavenging rate and hydroxyl radical scavenging rate as indicators to compare the advantages and disadvantages ofP.igniariusfermented mulberry black tea andE.cristatumfermented black tea. The statistical results are shown in Fig.1-5.

Note: * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01).The same as below.Fig.1 Comparison of polysaccharide content in mulberry black tea fermented by Phellinus igniarius and Eurotium cristatum

Fig.2 Comparison of polyphenol content in mulberry black tea fermented by Phellinus igniarius and Eurotium cristatum

Fig.3 Comparison of flavonoid content in mulberry black tea fermented by Phellinus igniarius and Eurotium cristatum

Fig.4 Comparison of DPPH· scavenging rate in mulberry black tea fermented by Phellinus igniarius and Eurotium cristatum

Fig.5 Comparison of hydroxyl radical scavenging rate in mulberry black tea fermented by Phellinus igniarius and Eurotium cristatum

From the data in Fig.1 to Fig.5, it can be found that high content of polysaccharides was produced after fermenting mulberry black tea byP.igniarius. Its polysaccharide content was 2.69 times that of the control mulberry black tea, reaching 0.410 mg/mL, and the corresponding hydroxyl radical scavenging rate was also higher with a hydroxyl radical scavenging rate of 60.31%, which is also consistent with the conclusion that polysaccharides are the main bioactive substances ofP.igniariusin previous studies[16]. However, the polyphenols in the mulberry black tea fermented byP.igniariuswere significantly decreased compared with the control mulberry black tea and the mulberry black tea fermented byE.cristatum, and the polyphenols in the mulberry black tea were decreased by 63.18% compared with the control mulberry black tea, only 30.55 μg/mL; compared with mulberry black tea, the flavonoid content decreased by 24.55% to 27.38 μg/mL, which may be due to the fact thatP.igniariusconsumes more lignin during the growth process, and the scavenging rate of DPPH· is related to the content of polyphenols in tea. Compared with the control mulberry black tea, the content of polysaccharides in mulberry black tea fermented byE.cristatumincreased by about 83.17% to 0.279 mg/mL, the content of polyphenols decreased by 14.75% to 70.74 μg/mL, and the content of flavonoids increased by 67.13%, up to 60.65 μg/mL.

3.2 Sensory analysisThe appearance and aroma of mulberry black tea were greatly affected by inoculation of different strains of bacteria. The comparative sensory analysis of three different mulberry black teas is shown in Table 1.

Table 1 Comparison of the sensory analysis between mulberry black tea fermented by Phellinus igniarius and Eurotium cristatum

As shown in Table 1, the mulberry black tea fermented byP.igniariuswas lighter in color, orange-red, slightly turbid, opaque, relatively light in aroma, but relatively pure and mellow in mouthfeel; mulberry black tea fermented byE.cristatumis was similar in color to unfermented mulberry black tea, had a brown-red color, and the tea soup was bright and the aroma was stronger than that of unfermented mulberry black tea.

4 Conclusions

We separately fermented mulberry black tea withP.igniariusandE.cristatumis to compare the functional components and antioxidant activities of the two fermented teas and the original mulberry black tea. The results showed that compared with the original mulberry black tea,P.igniariusfermented mulberry black tea produced more polysaccharides and had a higher hydroxyl radical scavenging rate; compared with the original mulberry black tea,E.cristatumis fermented mulberry black tea produced more flavonoids, and the production of polysaccharides also increased. However, the content of polyphenols in the two fermented teas was lower than that of the original mulberry black tea, which may be due to the consumption of lignin in the tea by the growth of the bacteria. Combined with sensory analysis,E.cristatumis fermented tea had the strongest tea aroma and clear color, whileP.igniariusfermented tea had a lighter tea aroma and a mellow mouthfeel, but the color was lighter and slightly turbid. In summary, the mulberry black tea fermented byP.igniariusandE.cristatumhas improved in different functional components, and has its own advantages in aroma and mouthfeel, so it has certain research value and market prospect.