Isolation of a novel characterized Issatchenkia terricola from red raspberry fruits on the degradation of citric acid and enrichment offlavonoid and volatile profiles in fermented red raspberry juice

Ying Jing, Ting Luo, Ying Tng, Sirui Chn, Hui Ni, Qih Chn,Xingshun Song, Yihong Bo,f, Zyun Dng*, Jinling Wng,f,*

a School of Forestry, Northeast Forestry University, Harbin 150040, China

b State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China,

c College of Food and Biological Engineering, JiMei University, Xiamen 361021, China

d College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China

e School of Life Sciences, Northeast Forestry University, Harbin 150040, China,

f Key Laboratory of Forest Food Resources Utilization of Heilongjiang Province, Harbin 150040, China

Keywords:

Red raspberry juice

Deacidification

Phenolic compounds

Volatiles

Issatchenkia terricola

A B S T R A C T

High content of citric acid in red raspberry juice leads to poor sensory experience. This study developed a feasible method to degrade citric acid in red raspberry juice using a novel characterized Issatchenkia terricola WJL-G4 isolated from red raspberry fruits. I. terricola WJL-G4 exhibited a potent capability of reducing the citric acid contents from (22.8 ± 0.08) g/L to (6.2 ± 0.02) g/L within 36 h fermentation, then completely depleted after 48 h. Furthermore, the contents of phenolic compound, including neochlorogenic acid,p-coumaric acid, raspberry ketone, and rutin significantly increased after 36 h fermentation. Fermentation increased total flavonoid contents in red raspberry juice, compared to that in control group. Volatile profiles exhibited to be enriched after fermentation, which contributed to the improvement of the juice taste. Our findings showed that I. terricola WJL-G4 can be applied in deacidification, enrichment of flavonoid compounds and volatile profiles in fermented red raspberry juice. .

1. Introduction

Red raspberry (Rubus idaeusL.), contains a variety of beneficial compounds such as raspberry ketone, ellagic acid, and anthocyanins,is cultivated throughout the world [1,2]. In recent decades, there has been a growing market and a considerable increase in the production of this small fruit [3,4]. Therapeutic effects of raspberry intake have been reported, such as eliminating the fatigue, improving gastrointestinal function, antibacterial action, and mitigating the progression of high fat diet induced behavioral dysfunction [3,5,6].Our previous findings indicated that raspberry ketone and ellagic acid in raspberry could ameliorate metabolic syndrome [7,8]. However,one of the main constraints for consuming and marketing raspberry fruits is the long-term storage, due to the high perishability of the fruits. Therefore, after harvest, approximately 68 4000 t of raspberry is processed each year [9], and raspberry juice is one of the major processed products of raspberry fruits [10].

The high content of citric acid in red raspberry juice, which results in poor sensory experience, limits its application in food industry.Commonly used methods to decrease the acidity of fruit juice include calcium salts precipitation, ion-exchange resins and electrodialysis.However, because of CO2release, precipitation using CaCO3is not recommended. An organoleptic characteristic is always shown in the juice processed with ion-exchange. Electrodialysis with homopolar membranes results in an increase in the sodium concentration [11].Therefore, developing new processing methods to adjust the pH value in red raspberry juice discloses new market and business opportunities.

The main organic acid in red raspberry juice is citric acid, which is about 20 g/L, accounts for more than 90% of the total acids in red raspberry juice [12]. Some studies had shown citric acid could be consumed as a sole carbon source by some yeasts in the fruits,includingIssatchenkia terricola[13,14]and thePichiagenus [15],which resulted in the acidity reduction. However, the degradation rates of citric acid by the yeasts mentioned above were low. For example, after fermentation, the degradation rate of citric acid byPichiagenus was from 12.3 g/L to 11 g/L [15]. Moreover, no biological acid-reducing method has been applied to reduce citric acid content in raspberry juice.

The aim of the present study was to evaluate the hypothesis that fermentation with yeasts from red raspberry fruits, could metabolize citric acid, and be applied to deacidify red raspberry juice. Changes in phytochemicals and volatile compounds of red raspberry juice during fermentation were investigated herein.

2. Materials and methods

2.1 Materials

All the standards for high performance liquid chromatography(HPLC), including rutin, quercetin, hyperin, chlorogenic acid, ellagic acid, isorhamnetin, raspberry ketone, epicatechin, catechinic acid,syringic acid, gallic acid, caffeic acid, luteolin, citric acid,L-malic acid, succinic acid,α-ketoglutarate, and et al. (HPLC grade) were obtained from Shanghai YuanYe Biotechnology (Shanghai, China).

Wallerstein Laboratory (WL) citric acid medium (WLC): WL nutrient liquid medium lacking a carbon energy source (0.5% yeast peptone, 0.4% yeast extract, 0.055% KH2PO4, 0.042 5% KCl,0.012 5% CaCl2, 0.012 5% MgSO4, 0.000 25% FeCl3, 0.000 25% MnSO4,m/V), supplemented with 20 g/L citric acid to be the unique carbon energy source. WLC was used to screen yeasts with the ability to degrade citric acid.

WL citric acid solid medium (WLCS): WLC added 2% agar.WLCS was used for yeasts isolation.

Yeast extract peptone dextrose (YPD) medium (1% yeast extract,2% yeast peptone, 2% glucose,m/V) was utilized to cultivate yeast strains.

YPD agar: YPD medium added 2% (m/V) agar.

Red raspberries of the cultivar “Autumn Bless”, one abundant raspberry species in Northeast China, were obtained from a local farm (Harbin, Heilongjiang, China) in Aug. 2019. The fruits with similar maturity were hand-picked from different trees and then snapfrozen. 40 kg of frozen red raspberries was mixed, transported to the laboratory in ice box and stored at -80 °C until use. Another 1 kg fresh hand-picked red raspberries was trasnsported to the laboratory in ice box and stored at 4 °C for yeast isolation next day.

2.2 Isolation of yeast strains from red raspberry fruits for citric acid degradation

2.2.1 Preliminary screening of yeast strains from red raspberry fruits

Two grams of fresh red raspberry fruits were placed into a conical flask containing 100 mL sterile saline and 8-12 sterile glass beads and the flask was shaken at 120 r/min for 20 min at room temperature in an incubator shaker (Shanghai Yiheng Scientific Instrument,Shanghai, China). The supernatant was harvested for yeast isolation after 2 h incubation.

Then a 5 mL aliquot of supernatant was added into a 250 mL flask with 100 mL of WLC, in which citric acid was the unique carbon energy source, then the flask was shaken at 120 r/min for 24 h at 28 °C, aimed to screen yeast strains with the ability to degrade high concentration of citric acid.

2.2.2 Isolation of selected yeast strains

The finally obtained yeasts solution was diluted and spreaded onto WLCS. The plates were then incubated for 2 days at 28 °C to obtain the single colony of selected yeast strains, followed by streak plate for the isolation of pure culture of selected yeast strains.

2.2.3 Isolated yeast strains on citric acid degradation

The finally obtained single yeast colonies were inoculated into 150 mL WLC, and statically cultured at 28 °C for 8 days. The initial inoculation was maintained at 8 × 107CFU/mL. Residual citric acid concentrations in WLC were measured daily by high performance liquid chromatography (HPLC) [16,17].

2.3 Yeast strain G4 identification

The screened yeast strains including G1, G2, G3, and G4 could tolerate high concentrations of citric acid. Strain G4, which exhibited highest capacity on citric acid degradation (degraded ～67% citric acid) in WLC, was identified.

2.3.1 Phenotypic identification

A single colony of isolated and purified strain G4 from 2.2.2 was streaked and inoculated onto YPD agar. The plates were cultured at 28 °C for 3 days, and the morphological characteristics of strain G4 were observed. After methylene blue staining or malachite green staining, the morphology and budding mode of the strain, as well as the shape and the number of ascospore in each ascus, were observed under a microscope (Olympus Guangzhou Industry, Guangzhou, China).

A single G4 colony was inoculated in YPD media. The media was statically cultured at 28 °C for 3 days, and the status of the yeast in the liquid medium was observed.

2.3.2 Molecular identification

The DNA of strain G4 was extracted using Yeast DNA kit (Sangon Biotech, Shanghai, China). The ITS1, 5.8S and ITS2 regions of rDNA gene were amplified by polymerase chain reaction (PCR) using forward primer ITS1 (5’-TCC GTA GGT GAA CCT GCG G-3’) and reverse primer ITS4 (5’-TCC TCC GCT TTA TTG ATA TGC-3’),according to Chavan et al. [18]. The conditions of PCR amplification were as followed: initial denaturation at 94 °C for 4 min; 30 cycles of denaturing at 94 °C for 45 s, annealing at 55 °C for 45 s and extension at 72 °C for 1 min; and a final extension at 72 °C for 10 min. The PCR products (2 μL) were separated by gel electrophoresis on 1% (m/V) agarose. After electrophoresis at 150 V for 20 min, gels were visualized under UV light and photographed by Alpha Innotech (Alpha, San Leandro, CA, USA). Sizes were estimated by comparison against a DNA size marker.

PCR products were purified using PCR purification kit (Qiagen,Germantown, MD, USA). The sequence of final PCR product was compared with these available in the GenBank database through the search tool (BLAST).

Molecular Evolutionary Genetics Analysis (MEGA) Version 7 software was used to conduct phylogenetic trees, inferring evolution of the identified strain G4.

2.4 Preparation of red raspberry juice

To keep the consistency of sampling, at least 6 kg red raspberry fruits were milled after natural thawing. After an enzymatic treatment(pectinase, 40 000 U/g, enzyme : substrate = 0.02 : 100) at a temperature of 50 °C for 2 h, the obtained red raspberry pulp was incubated at 90 °C for 5 min for enzyme deactivation, followed by filtration using an 8-layer gauze. More than 3 L red raspberry juice was obtained,vacuum degassed and pasteurized at 80 °C for 15 min, and cooled down for future experiment, including the determination of organic acids,phenolic compounds, total anthocyanin et al. (sample size was 3).

2.5 The deacidification ability of isolated strain G4 from red raspberry fruits on fermented red raspberry juice

The yeast strain G4 which possessed the highest ability on citric acid degradation, based on the results from 2.2.3, was inoculated at a concentration of 8 × 107CFU/mL in the red raspberry juice. Yeast strain G4 was cultured with or without shaking (120 r/min, optimized speed, data not shown) at 28 °C for 72 h. The red raspberry juice without yeast inoculation was set as the control group. The changes of pH value in red raspberry juice were measured daily.

2.6 Organic acids determination of red raspberry juice fermented with strain G4

Based on the results from 2.5, the deacidification ability of strain G4 was higher when fermentated with shaking compared to without shaking. Therefore, the following fermentation were all done with shaking at a speed of 120 r/min.

Supernatant of control and fermented red raspberry juice was harvested after centrifugation at 4 000 r/min for 15 min. After filtered through a 0.22 μm membrane, organic acids, including citric acid,L-malic acid, succinic acid, andα-ketoglutarate were measured using HPLC. Organic acids in the samples were detected using an Agilent ZORBAX Eclipse Plus C18column (250 mm × 4.6 mm) (Agilent Technologies Inc., Santa Clara, CA, USA). Mobile phase was consisted of 0.5% KH2PO4(m/V, pH 2.5, solvent A) and methanol(solvent B). The ratio of A : B was 97 : 3 (V/V). The rate for mobile phase was 0.7 mL/min. Volume injection was 10 μL and the column temperature was 35 °C. Wavelength for the detection was 210 nm.The elution time was 10 min.

Identification of the compounds was done by comparison of their retention time with those of the standards.

2.7 The changes of total phenolic contents in red raspberry juice during fermentation with strain G4

Total phenolic contents in the red raspberry juice fermented with strain G4 were measured using Folin-Ciocalteu method, as described previously [19]. Brie fly, 1 mL of red raspberry juice was mixed with 1 mL of Folin-Ciocalteu reagent and 1 mL of 7.5% (m/V) sodium carbonate. The mixture was brought to 5 mL using distilled water and was allowed to stand at room temperature for 30 min. Finally,the absorbance was measured at 765 nm against a blank. Phenolic contents were estimated from a standard curve obtained from gallic acid. Total phenolic contents were expressed as mg gallic acid equivalent per milliliter red raspberry juice (mg GAE/mL). Samples were read in triplicate.

2.8 The changes of total anthocyanin contents in red raspberry juice during fermentation with strain G4

pH differential method was used to measure total monomeric anthocyanin, which is a spectrophotometric method based on the anthocyanin structural transformation that occurs with a change in pH(colored at pH 1.0 and colorless at pH 4.5) [20].

Mis the molecular weight.DFis the dilution factor.εis the molar absorptivity.A=(A510nm·pH1.0-A700nm·pH1.0)-(A510nm·pH4.5-A700nm·pH4.5).

2.9 The changes of total flavonoid contents in red raspberry juice during fermentation with strain G4

The total flavonoid contents were evaluated using aluminium nitrate nonahydrate [19,21]. The total flavonoid concentrations were calculated from rutin calibration curve and expressed as mg Rutin/mL red raspberry juice (mg Rutin/mL).

2.10 HPLC quantitative analysis of phenolic compounds in red raspberry juice during fermentation with strain G4

Supernatant of fermented red raspberry juice was harvested after centrifugation at 4 000 r/min for 15 min. After filtered through a 0.22 μm membrane, phenolic compounds in the samples were separated using an Agilent ZORBAX Eclipse Plus C18column (250 mm × 4.6 mm). Mobile phase consisted of methanol (solvent A) and 0.02% formic acid in water(m/V, solvent B). The rate for mobile phase was 0.8 mL/min. Volume injection was 10 μL and the column temperature was 35 °C. Wavelength was 280 nm. The elution was conducted as follows:

0-5 min, programmed from 0% A to10% A; 5-10 min, from 10% A to 20% A; 10-20 min, from 20% A to 35% A; 20-35 min, from 35% A to 40% A; 35-40 min, from 40% A to 75% A; 40-45 min, from 75% A to 10% A.

Identification of the compounds was done by comparison of their retention time with those of the standards. Stock solutions of standards were prepared in methanol: dimethyl sulfoxide (DMSO)(90 : 10,V/V).

2.11 HS-SPME-GC-MS analysis of volatile compounds in red raspberry juice during fermentation with strain G4

Volatile compounds in fermented red raspberry juice were analyzed using headspace solid-phase microextraction and gas chromatograph-mass spectrometry (HS-SPME-GC-MS), according to a previous method with slight modifications [22].

Volatiles were extracted using SPME device equipped with a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS)fiber (Supelco, Bellefonte, PA, USA). Red raspberry juice (5 mL)and 1 g of sodium chloride were sealed in a 20 mL headspace vial,and equilibrated at 50 °C water bath for 10 min. Then the volatile compounds were headspace extracted by fiber at 50 °C for 40 min [22].

The volatile compounds were then desorbed for 2 min [22,23]into the injector of an Agilent 5975 GC/MS (Santa Clara, CA,USA) equipped with a DB-WAX fused silica capillary column(0.25 mm × 30 m i.d., film thickness 0.25 μm). The mass spectral ionization temperature was set at 230 °C. The mass spectrometer was operated in the electron impact ionization mode (70 eV), and mass spectra were acquired by scanning over the mass/charge (m/z)range of 50-500 AMU/sec. The injector temperature was 250 °C. The column temperature was programmed from 40 °C (held for 3 min) to 160 °C (held for 2 min) at a rate of 3 °C/min, and finally increased to 220 °C (held for 3 min) at a rate of 8 °C/min [24].

Volatile compounds were tentatively identified if their match quality was ≥ 80 by comparing their mass spectra with those of reference compounds in the data system of the Wiley library and NIST Mass Spectral Search Program (ChemSW Inc., NIST 98 Version Database) connected to an Agilent 5975 mass spectrometer.All analyses were performed in triplicate. The results were expressed as the mean ± standard deviation of three samples.

2.12 Sensory evaluation analysis of red raspberry juice

Pasteurization was used before sensory evaluation. Totally 11 red raspberry juice (5 fermented-12, 24, 36, 48, and 60 h after fermentation, 6 unfermented-0, 12, 24, 36, 48, and 60 h of control group) were sensory evaluated by ten trained judges selected from students and teaching staff of the department. The tasters were given an evaluation form. The details of the grade were shown in Tables 1 and 2. Grades (0-20 or 30, whereas 0-minimal, 20 or 30-maximal)were carried out based on the following sensory parameters: the samples were evaluated by color (purity, browning, typicality and overall sensation), flavor (purity, typicality, intensity and overall sensation), taste (acidity, sweetness, bitter-sweet taste, harmonious taste, astringency, mouth-feel and overall sensation), and organization status. Each taster assessed 5 mL of juice, which was served in 50 mL plastic cups. Sensory evaluation was carried out in batches with appropriate pauses.

2.13 Statistical analysis

All analyses were run in triplicate and were expressed as mean ±standard deviation. Statistical analysis was done by using SPSS 24.0 Software (SPSS Inc., Chicago, IL, USA). Differences between means were analyzed by one-way ANOVA followed by LSD (P＜ 0.05).

3. Results and discussion

3.1 Isolation and identification of yeast strains with high deacidification activity from red raspberry fruits

Citric acid, a dominant organic acid in red raspberry juice,accounts for more than 90% of the total acids in red raspberry juice. In order to obtain a highly efficient yeast strain for citric acid degradation in red raspberry juice, high citric acid content was used to incubate yeasts isolated from red raspberry fruits. There were 4 yeast strains, including G1, G2, G3, and G4 tolerated high concentration(20 g/L) of citric acid. As shown in Supplementary Fig. 1, these four strains showed different abilities on decreasing citric acid. Strain G4 behaved the highest deacidification capacity. The reduction rate was high in the first several days and then reached a plateau 7 days after incubation. After 8 days of fermentation with G4, the total amount of citric acid was declined from 20 g/L to (6.55 ± 0.72) g/L. The degradation rate was ～67 % in this condition. Therefore, yeast strain G4 was used to perform the following studies.

Strain G4 was then identified on the basis of colony characteristics, morphology, and molecular techniques.

The isolated strain G4 were first identified by studying specific morphological, biochemical and physiological characteristics. As shown in Supplementary Fig. 2A, the morphology of the yeast, 3 days after growth on YPD agar, the colony of G4 displayed creamy,smooth and butyrous. Ovoidal to elongate shapes of the yeast were observed. A protuberance from the cell wall was displayed, as shown in Supplementary Fig. 2B, G4 was reproduced by budding. Ascus housed one to four ascospores and two spores per ascus were most typical (Supplementary Fig. 2C). After incubation for 3 days at 28 °C,pellicle was formed on the surface of the culture medium.

Fig. 2 Organic acid contents in fermented and control red raspberry juice. The changes of organic acid contents in red raspberry juice were measured every 12 h.(A) citric acid, (B) L-malic acid, (C) succinic acid, (D) α-ketoglutarate. Samples labeled with the same letter are not significantly different from each other, P ＜ 0.05(mean ± S.D., n = 3).

Furthermore, The ITS1-5.8S-ITS2 region of strain G4 was obtained by amplification with ITS1 and ITS4 fungal primers. Based on the ITS sequence, phylogenetic trees were conducted to infer the evolution of the identified strain G4 (Supplementary Fig. 3).

Fig. 3 Total phenolic, anthocyanin, and total flavonoid contents of fermented andcontrol red raspberryjuice.The changes of total phenolic contents(A), anthocyanin contents (B), and total flavonoid contents (C)in redraspberry juice were measured every12 h. Samples labeledwith the same letter are not signif i cantly different from each other, P ＜0.05 (mean± S.D., n =3).

The combined phenotypic and molecular approach enabled the identification of strain G4 belonging to theI. terricola. We named it asI. terricolaWJL-G4 (Chinese patent No. 2019113316700) and stored it at China General Microbiological Culture Collection Center(CGMCC), No. 18712.

3.2 The deacidification ability of isolated yeast stain G4 on red raspberry juice

Fig. 1 showed that without shaking,I. terricolaWJL-G4 had a slight effect on the pH values of red raspberry juice during 72 h fermentation. However, shaking at a speed of 120 r/min significantly increased the pH values of red raspberry juice, from 3.08 ± 0.03 to 4.84 ± 0.10 after 72 h.

Table 1Sensory evaluation standards.

Table 2Sensory evaluation test results.

Based on this finding and a study of shaking speed optimization(data not shown), we performed the fermentation usingI.terricolaWJL-G4 under a constant shaking of 120 r/min in the following studies.

3.3 Organic acid changes of red raspberry juice

We further evaluated the regulation of organic acid contents during the fermentation process using HPLC, including citric acid,L-malic acid, succinic acid, andα-ketoglutarate, which are the main organic acids involved in the tricarboxylic acid cycle (TCA cycle).As shown in Fig. 2, the most common organic acid was citric acid,followed by succinic acid andL-malic acid, however,α-ketoglutarate accounted for the least. After 48 h of fermentation, citric acid in the red raspberry juice was almost completely depleted from (22.8 ±0.08) g/L.L-malic acid contents declined from (1.28 ± 0.02) g/L to(0.65 ± 0.002) g/L during 72 h fermentation. Succinic acid andα-ketoglutarate contents increased from 0 h to 36 h and 24 h,respectively, and then declined.

α-Ketoglutarate and succinic acid are downstream products of citric acid in TCA cycle.L-malic acid, an intermediate product of glyoxylate cycle, can be generated from citric acid as well. However,as the depletion of citric acid during the fermentation process,α-ketoglutarate, succinic acid, andL-malic acid were utilized as the carbon energy source, which resulted in the reduction ofα-ketoglutarate, succinic acid, andL-malic acid at a later stage, for example 48 h after fermentation.

Cássio and Leao [25]found thatC. utilis, a yeast species,possessed carboxylic acid transporters for citric acid uptake, therefore,utilized citric acid as an important energy source via the TCA cycle.Similarly, in order to utilize citric acid as the sole carbon source,I. terricolaWJL-G4 is required to express carboxylic acid transporters and essential enzymes for TCA cycle or related metabolic pathway,however, it is imperative to investigate the expression of carboxylic acid transporters and enzymes required for citric acid degradation inI. terricolaWJL-G4 in future studies.

3.4 The changes of total phenolic contents, anthocyanin contents, and flavonoid contents in red raspberry juice

WhetherI. terricolaWJL-G4 fermentation altered the total phenolic contents was then tested. Surprisingly, with a shaking speed of 120 r/min, total phenolic contents declined from(1.33 ± 0.09) mg GAE/mL at baseline to (0.99 ± 0.09) mg GAE/mL after 72 h fermentation. The reduction of total phenolic contents might be attributed to degradation and hydrolysis of the phenolic compounds during incubation at room temperature [26]. However,no significant difference of phenolic contents was observed between control and fermented red raspberry juice except 36 h (Fig. 3A).

Two main phenolics in red raspberry juice, including anthocyanins and flavonoids were detected during fermentation process (Figs. 3B and 3C). Consistently, anthocyanins were significantly decreased after fermentation. However, no significant difference of anthocyanins between control and fermented groups was observed after 12, 24, 36 and 60 h of fermentation. Therefore, we might draw a conclusion that fermentation exhibited no significant effect on total phenolic contents and total anthocyanin contents in red raspberry juice.

On the other hand, interestingly, fermentation increased total flavonoid contents in red raspberry juice, compared to the contents in control group. After 72 h, the total flavonoid contents were(0.85 ± 0.02) and (0.57 ± 0.06) mg Rutin/mL in fermented and control groups, respectively.

3.5 Phenolic compounds in red raspberry juice

Furthermore, the specific phenolic compounds and their contents in the fermented and nonfermented (control group) red raspberry juice were measured by HPLC analysis. Phenolic acid, such as cryptochlorogenic acid, the amount was enriched after 48 h of fermentation withI. terricolaWJL-G4, from (3.82 ± 0.59) mg/L in the control group to (4.21 ± 0.65) mg/L in fermented group (Table 3).Notedly, after fermentation for 36, 48, and 60 h, the contents of neochlorogenic acid andp-coumaric acid robustly increased.

Table 3Phenolic prof i les of fermented andcontrol red raspberryjuice.

Flavonoids including raspberry ketone, isostatin, hypericin,rutin, naringin, quercetin, luteolin, epicatechin, arbutin, and kaempferol were measured. Raspberry ketone, the secondary metabolite of raspberry, was significantly increased after 36,48 and 60 h fermentation. After 48 h, raspberry ketone contents in fermented and control group were (19.48 ± 2.65) and(11.27 ± 0.01) mg/L, respectively. Raspberry ketone has attracted a lot attention nowadays and has been reported to ameliorate the symptoms of obesity. Luo et al. [8]found that the consumption of raspberry ketone reduced high-fat diet induced mice body weight gain and hepatic lipid accumulation. Wang et al. [27]reported that raspberry ketone protected rats against nonalcoholic steatohepatitis.Therefore, after fermentation, the function of red raspberry juice on obesity might be improved. Also, rutin was robustly increased after fermentation withI. terricolaWJL-G4. Consistently, after fermentation, quercetin was enhanced, compared to control group.

Polyphenols in fruit juice are usually found glycosylated with sugar [28]. Some organisms were capable of producingβ-galactosidase during the fermentation, which could result in the release of phenolic compounds from the bounded sugar [29]. Therefore, the contents of some polyphenols in red raspberry juice were increased after fermentation in our study. Furthermore, actually, the increase in the antioxidant activity of fermented anthocyanin glycosides by some micro-organisms was reported by ávila et al. [30]. Other researchers found the increase of bioavailability of phenolic compounds and antioxidant activity of fruits juice, such as pomegranate juice [31].

3.6 The changes of volatile compounds of red raspberry juice

Volatile compounds play a key role in the formation of the wellrecognized and widely appreciated raspberry aroma. Metabolism of phenolic compounds during fermentation has been reported to lead to generation of compounds that impact flavor [32,33]. To test the hypothesis in the study, we measured the volatile compounds and performed sensory evaluation analysis of red raspberry juice fermented withI. terricolaWJL-G4.

The results of the volatiles analysis of the red raspberry juice with and without fermentation were shown in Table 4.

Table 4Volatile compounds of fermentedand control red raspberry juice.

Benzaldehyde, which provides an oily taste and found in unfermented red raspberry juice was not identified after fermention withI. terricolaWJL-G4.

Fermentation withI. terricolaWJL-G4 robustly enhanced the relative contents of phenylethyl alcohol in red raspberry juice.Phenethyl alcohol, provides a rose-like flavor, has been reported to be produced by yeasts during alcoholic fermentation by conversion ofL-phenylalanine presented in the medium or byde novosynthesis from sugar substrates [34].

Some esters, such as ethyl octoate, and ethyl decanoate were undetected in unfermented red raspberry juice, however, the amounts were increased in fermented red raspberry juice. The reason was that medium chain fatty acid ethyl esters were largely formed during the fermentation by the enzymatic condensation of fatty acyl-CoA with ethanol, where fatty acyl-CoA was produced from acetyl-CoA mainly comes from the oxidative decarboxylation of pyruvate [35]. Esters are responsible for sweet and fruity odor. Also, esters often act in synergy with other compounds to affect beer flavor in concentrations well below their individual threshold values [36]. Therefore, a stronger fruity and weaker floral aroma was expected to show in fermented red raspberry juice.

3.7 Sensory evaluation analysis of red raspberry juice

A sensory evaluation test was performed to determine the quality and consumer acceptance of fermented red raspberry juice. Short term fermentation significantly improved the taste of red raspberry juice. For example, 36 h after fermentation, the taste score of juice was robustly increased compared to the juice without fermentation(26.40 ± 1.78 vs. 22.90 ± 2.23). However, red raspberry juice with a long-time fermentation (48 and 60 h) usingI. terricolaWJL-G4 showed a lower overall consumer acceptance, compared to the juice without any yeasts inoculated or the juice with relative short time fermentation. In detail, the scores offlavor and taste were found to be significantly decreased, since based on Fig. 2, citric acid robustly decreased after 48 and 60 h of fermentation. Citric acid is frequently used as a food additive to provide acidity and sour taste. The flavor quality of fruits and fruit juice is largely determined by the sugaracid ratio [37]. During short-time fermentation, no significant impact of flavor and taste properties was detected, compared with unfermented juice.

4. Conclusion

Fermentation with a new identifiedI. terricolaWJL-G4 can be applied in deacidification of red raspberry fruits and enrich the profiles of flavonids and volatile compounds. It is expected that this work will expedite research on the production of highquality raspberry juice with beneficial physicochemical properties,functionality and good sensory characteristics.

5. Future work

Whole genome sequencing can be used in the future to investigate genes which are responsible for the conversion of phenolic especially flavonoid consistituents and volatile compounds in fermented red raspberry juice. On the other hand, the mechanism of howI. terricolaWJL-G4 degrades citric acid will be elucidated in the future.

Conflict of interest

The authors have no conflict of interest to report.

Fundings

This work was supported by Heilongjiang Tongsheng Food Technology Co., Ltd., and “the Fundamental Research Funds for the Central Universities” [grant number 2572018BA07 and 2572018CG02]; and “Applied Technology Research and Development Project of Harbin Science and Technology Bureau”[grant number 2017RAYXJ012].

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://doi.org/10.1016/j.fshw.2022.03.029.