Mulberry anthocyanin biotransformation by intestinal probiotics

Accepted Manuscript Mulberry anthocyanin biotransformation by intestinal probiotics Jing-Rong Cheng, Xue-Ming Liu, Zhi-Yi Chen, You-Sheng Zhang, Ye-Hui Zhang PII: DOI: Reference:

S0308-8146(16)31054-8 http://dx.doi.org/10.1016/j.foodchem.2016.07.032 FOCH 19493

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

30 November 2015 1 June 2016 5 July 2016

Please cite this article as: Cheng, J-R., Liu, X-M., Chen, Z-Y., Zhang, Y-S., Zhang, Y-H., Mulberry anthocyanin biotransformation by intestinal probiotics, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem. 2016.07.032

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Food Chemistry Mulberry anthocyanin biotransformation by intestinal probiotics

Jing-Rong Chenga,*, Xue-Ming Liua,*, Zhi-Yi Chena, You-Sheng Zhanga, Ye-Hui Zhanga

a

Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences, Key Laboratory of Functional Foods, Ministry of Agriculture, Guangdong Key Laboratory of Agricultural Products Processing. Guangzhou 510610, PR China

*Corresponding author. Address: 133 Yihenglu, Dongguanzhuang, Tianhe District, Guangzhou 510640, PR China Tel. /fax:+86 020 37203765(J.R.Cheng); +86 020 37203765 (X.M. Liu) E-mail address: [email protected]; [email protected]

1

Abstract This study was designed to evaluate mulberry anthocyanins bioconversion traits for intestinal probiotics. Five intestinal beneficial bacteria were incubated with mulberry anthocyanins under anaerobic conditions at 37

o

C, and bacterial

β-glucosidase activity and anthocyanin level were determined. Results demonstrated that all strains could convert mulberry anthocyanins to some extent. With high β-glucosidase production capacity, S. thermophiles GIM 1.321 and L. plantarum GIM 1.35 degraded mulberry anthocyanins by 46.17% and 43.62%, respectively. Mulberry anthocyanins were mainly biotransformed to chlorogenic acid, crypto-chlorogenic acid, caffeic acid, and ferulic acid during the anaerobic process. Non-enzymatic deglycosylation of anthocyanins also occurred and approximately 19.4% of the anthocyanins were degraded within 48 h by this method.

Keywords: β-Glucosidase; Mulberry; Anthocyanins; Bioconversion; Intestinal beneficial bacteria; Phenolic acids

2

1. Introduction Anthocyanins are natural colorants which have aroused a growing interest due to their extensive range of colours, innocuous and beneficial health effects (Araceli, Lourdes, Ma, Jose, & Carlos, 2009). They are considered potential food additives and/or synthetic pigment substitutes. In vitro and in vivo studies during the past decade have found that anthocyanins possess antioxidant, anti-inflammatory, vasoprotective, antineoplastic, chemo- and hepato-protective activities and they have been widely used for therapeutic purposes (Ghasemzadeh, Jaafar, & Karimi, 2012; Luo, Zhang, Yan, Tang, Ding, Yang, et al., 2011). Anthocyanins occur in all tissues of higher plants including leaves, stems, roots, flowers and fruits. Large amount of plant species, such as grape, elderberries, red cabbage, blood orange, black chokeberry, sweet potato, etc., are classified as potential sources of anthocyanin (Bridle & Timberlake, 1997). We previously showed that mulberry fruit, is also a source of anthocyanins such as cyanidin3-glucoside and cyanidin3-rutinoside (Liu, Xiao, Chen, Xu, & Wu, 2004). Among the great efforts done to exploiting the anthocyanins pharmacological activity and their application in food industry, their mechanisms of metabolic pathway has received increasing attention over the recent years (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009). Due to sugar-based connection, anthocyanins are not readily absorbed by the digestive tract, but hydrolyed to aglycones by the intestinal bacterial glycosidase or further degrade into phenolic acids (Liang, Wu, Zhao, Zhao, Li, Zou, et al., 2012). As such the 3

bioavailability of anthocyanins is considered very low based mainly on the measurement of original anthocyanins and conjugated metabolites, glucuronidated, and sulfated anthocyanins in plasma or urine (Liang, et al., 2012; Manach, Williamson, Morand, Scalbert, & Remesy, 2005). Related studies reported very low absorption and urinary excretion of intact anthocyanidin glycosides between 0.016% and 0.110% of the dosage in humans and in different animal models (Bub, Watzl, Heeb, Rechkemmer, & Briviba, 2001; Netzel, Strass, Janssen, Bitsch, & Bitsch, 2001). More recently, it has been established that intestinal microflora play a key role in the metabolism of anthocyanin. After ingestion, the anthocyanins are hydrolyzed by intestinal glycosidase and the resulting aglycones are further metabolized in the large intestine into other metabolites such as protocatechuic, gallic, syringic and vanillic acids (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009; Forester & Waterhouse, 2008). It is reported that anthocyanins could be degraded by gut microflora giving rise to the formation of other breakdown metabolites, which could also contribute to health effects associated with anthocyanin (Hidalgo, Martin-Santamaria, Recio, Sanchez-Moreno, de Pascual-Teresa, Rimbach, et al., 2012). For example, protocatechuic acid a isopropyl ester, reduced plasma TNF-α, Nitric Oxide and hepatic malondialdehyde levels in a mouse model of septic shock induced by lipopolysaccharide and D-galactosamine (Yan, Jung, Hong, Moon, Suh, Kim, et al., 2004). Gallic acid could be an effective protector against monocyte recruitment in inflammatory vessels and might be instrumental in preventing atherosclerotic lesion development (Hidalgo et al., 2012). However, microbial 4

biotransformation efficacy of anthocyanins by intestinal bacterial types involved in anthocyanin conversion to bioactive forms is not entirely known. Structurally, anthocyanins are polyhydroxy or polymethoxy derivatives of 22-phenyl-benzopyrylium often with β-3-O-glycosidic or β-3,5-O-diglycosidic bonds (Markham, Geiger, & Harborne, 1994). Herein, hydrolysis of anthocyanin glycoside is proposed as the first step for subsequent bacterial degradation. Intestinal bacteria, equipped with a vast array of enzymes, play important roles in anthocyanin bioconversion. Some of the ingested flavonoids are directly absorbed as glycosides through the epithelium sugar transporters or hydrolyzed by intestinal mucosal enzymes and absorbed passively, and then undergo conjugation in the ileal epithelium or in the liver (Aura, 2008). Recently, several in vitro incubation experiments have been designed to investigate flavonoid metabolism by intestinal microorganisms (Flores, Ruiz del Castillo, Costabile, Klee, Bigetti Guergoletto, & Gibson, 2015; Szwajgier & Jakubczyk, 2010). The identification and quantification of the metabolites confirmed that intestinal microflora had an enormous hydrolytic potential by cracking

glycosidic

bonds (Keppler

and

Hunpf,

2005).

Anthocyanin

monoglucosides and diglucosides can be de-glycosylated by colonic microflora in a 2 h period (Aura, Pilar, Karen Anne, Gary, Kirsi-Marja, Kaisa, et al., 2005; Keppler & Humpf, 2005). Hereafter, aglycons appear transiently, and are further degraded into phenolic acids corresponding to anthocyanidin ring-B (Aura, et al., 2005). To date, to the best knowledge of the authors, a limited number of studies are focused on mulberry anthocyanins biotransformation. 5

Lactobacillus sp., bifidobacterium and Streptococcus sp., are the predominant members of the intestinal beneficial microflora, and they are widely used in probiotic products (Meurman, Antila, Korhonen, & Salminen, 1995). The ability of lactobacilli and bifidobacteria to modify the gut microbiota and reduce the risk of cancer are in part due to their ability to decrease β-glucuronidase and carcinogen levels (Hosoda, Hashimoto, He, Morita, & Hosono, 1996). A relatively large volume of literatures support the proposition that probiotics positively modulate intestinal bacterial populations and several therapeutic mechanisms, including stimulation of immunity, competition for limited nutrients, inhibition of epithelial and mucosal adherence, inhibition of epithelial invasion and production of antimicrobial substances, have been proposed (Rolfe, 2000). Studies have revealed that most intestinal microflora possess β-glucosidase activity and participate in the hydrolysis of plant β-glycosides (Jeon, Ji, & Hwang, 2002; Otieno, Ashton, & Shah, 2005), while limited studies report about their potential for anthocyanin biotransformation. Providing anthocyanins were efficiently transformed to compounds with high bioavailability and pharmacological activity by intestinal microflora, anthocyanin based probiotic food would open up a new perspective to anthocyanins application in health food. In the present study, five intestinal beneficial bacteria were employed to investigate bacteria–anthocyanin interactions and

the mulberry anthocyanin

bioconversion using in vitro batch culture systems. In addition, their β-glucosidase production capacity for converting mulberry anthocyanins into other compounds was also examined. 6

2. Materials and methods 2.1 Chemicals Cyanidin-3-glucoside (C3G) and Cyanidin-3-rutinoside (C3R) were purchased from Polyphenols AS (Sandnes, Norway); cryptochlorogenic acid, chlorogenic acid, caffeic acid, and ferulic acid purchased from Merck (Darmstadt, Germany). 2.2 Mulberry anthocyanin preparation Mulberry fruits were obtained from South China Mulberry Germplasm Resource Garden in Baiyun Agricultural Experimental Base of the Guangdong Academy of Agricultural Sciences (Guangdong, China). The mulberry anthocyanins were treated and purified by X-5 resin according to method reported by our previous study (Liu, Xiao, Chen, Xu, & Wu, 2004). 2.3 Bacterial strains and their growth conditions Lactobacillus acidophilus GIM 1.83, Lactobacillus bulgaricus GIM 1.155, Bifidobacterium animalis GIM 1.169, Lactobacillus plantarum GIM 1.35 and Streptococcus thermophiles GIM 1.321 were purchased from Guangdong Institute of Microbiology. Cultures were maintained at -80 oC in MRS broth (Sigma-Aldrich, USA) supplemented with glycerol (40% v/v). Before the experimental phase, cultures were propagated twice in MRS broth at 37 oC for 24 h to obtain bacteria at the end of the exponential phase/beginning of the stationary phase. For experimental runs, the basal growth mediums for all strains were MRS broth. A stock solution of purified mulberry anthocyanins was prepared, sterilized with a filter and added to the growth medium to reach a final concentration of about 80 mg 7

L-1. Aliquots of MRS medium with equal amounts of anthocyanin were prepared as matrix blank samples. For all runs, inoculation was down at 2% and fermentation was performed at 37 oC under anaerobic conditions, without shaking, until the late stationary phase. At several time points the fermentation broth were sampled for measuring growth, anthocyanin degradation and other indexes. For the anthocyanin concentration analyses, an aliquot was mixed with 1% (v/v) phosphoric acid (85%) to prevent further degradation. All experiments were performed in triplicate. Mean values and standard deviations were presented. 2.4 β-Glucosidase activity assay For the liquid samples, cells were harvested by centrifugation (10,000×g, 15 min, 4 oC) and washed twice with 50 mM sodium phosphate buffer, pH 6.5. To obtain the whole-cell concentrates, harvested cells from different cultures were resuspended in the same buffer to an OD600 of ~1.0 and immediately tested for β-glucosidase activity according to the method reported by other’s study (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009). One unit of enzyme activity was defined as the amount of β-glucosidase that released 1µmol of p-nitrophenol from the substrate per min. 2.5 Total anthocyanin content The total anthocyanin content was determined using the pH differential method (Pavlovic, Dabic, Momirovic, Dojcinovic, Milojkovic-Opsenica, Tesic, et al., 2013). An 1800 UV spectrophotometer (Shimadzu, Japanese) with a 1 cm path length was used for spectral measurements at 520, and 700 nm. Pigment content was calculated 8

as cyanidin-3-glucoside, using an extinction coefficient of 29 600 L cm-1 mg-1 and molecular weight of 449.2 g mol-1. 2.6 Analysis and identification of anthocyanins and their metabolites Analysis of anthocyanin glycoside degradation and the formation of anthocyanin metabolites was carried out as described by others (Aura, et al., 2005; Liu, Xiao, Chen, Xu, & Wu, 2004). Samples were filtered through a 0.22µm filter and injected into the HPLC system. The HPLC system was equipped with XBridgeTM C18 column (5µm; 200Å; 4.6×250 mm) which was set thermostatically at 25oC. Solvents used were aqueous 10% formic acid (A) and HPLC-grade acetonitrile (B) at a flow rate of 0.8 mL/min. Starting with 6% A, the gradient was 9% B from 0 to 7 min, 11% B from 7 to 18 min, 14% B from 18 to 21 min, 22% B from 21 to 26 min, 30% B from 26 to 30 min and from 30 to 34 min 6% B. Detection wavelengths were 254 and 520 nm and samples were analyzed in triplicate. Anthocyanins were detected at 520 nm and their peak areas were referred to calibration curves obtained with cyanidin-3-glucoside or cyanidin-3-rutinoside (Fig.S1). Quantification of anthocyanins was accomplished by external calibration, using standard curves generated by a series of standard solutions between 0.0001 and 0.1 mg/mL of each anthocyanin. Areas of identified phenolic acids were compared with calibration curves prepared with the corresponding standard acids, wherever possible. 2.7 Statistical analysis All the analyses were performed in triplicate and results were presented as mean ± standard deviations of three independent incubations. 9

3. Results and discussion 3.1 Bacterial growth profiles Growth of the strains reflected their potential to transform anthocyanins indirectly. Researcher pointed out that the anthocyanin transformation capacity of bacteria is closely related to their growth cycle, and the highest transformation activity occurred at the late exponential phase (Otieno, Ashton, & Shah, 2005). In this experiment, OD600 value with the corresponding pH was adopted to monitor strain growth and mulberry anthocyanin biotransformation process (Fig. 1). Even though there were some fluctuations, cells showed logarithmic growth over the first 24 hours. Subsequently, growth slowed down between 24 and 36 h of incubation, which reflected a change from exponential to stationary growth phases, and then OD600 decreased between 36 and 48 h of incubation. It is observed from strain growth change curves that anthocyanins inhibited the proliferation of these strains since their OD600 value were relatively lower than their control. Apart from L. plantarum GIM 1.35 and S. thermophilies GIM 1.321, growth of most strains appeared to be slowing and the lag phase was pronounced between 0-8 h. A typical example of the suppression effect could be found for B. animals GIM 1.169, for which the OD600 value was kept at a low level (OD600 < 0.5) until 36 h by anthocyanins (Fig.1). This could be explained from two aspects. Firstly, the acidic solution of mulberry anthocyanins led to an unsuitable pH for the strains (Fig. 1) the hindering the test strains capacity to take up nutrients (Stead, 1993; Tobias, 1968). Secondly, anthocyanins belong to flavonoids, of which phenolic hydroxyl groups 10

could react with mycoproteins or enzymes through hydrogen bonding, leading to cytoplasm pyknosis and disintegration (Shi, Bassa, Gabriel, & Francis, 1992). Stead (1993) reported a similar inhibition effect of ferulic acid on the growth of L. collinoides in acid tomato broth (pH 4.8). The pH decrement was accompanied by culture growth during whole fermentation process. At the end of fermentation, pH changes for most cultures were not significant (p > 0.05). However, it was interesting that anthocyanins showed a prominent negative effect on B. animals GIM 1.169, of which the final pH was 3.85 while that of control was 4.35. With anthocyanins, its exponential phase delayed until 24 h and kept for an extended period (24 h-48 h). Strain of L. plantarum GIM 1.35 and S. thermophilies GIM 1.321 displayed the best growth performance with mulberry anthocyanins since little change of the growth cycle was evident between these two runs. The two strains exhibited significant proliferation in the first 24 h, of which OD600 values were 2.41±0.09 and 2.38±0.11, respectively.

3.2 β-Glucosidase production capacity of intestinal probiotics Generally, intestinal microflora possess β-glucosidase, β-galactosidase and β-galactosidase, which play important roles in hydrolyzing isoflavone glucosides to bioavailable aglycones forms (Otieno, Ashton, & Shah, 2005). Since the β-glucosidic linkage existed in all the investigated anthocyanins, we investigated the β-glucosidase activity of these five strains. All tested strains were positive for this activity over a 48-h incubation period at 37 oC and the results were presented in Fig. 2. There was a significant difference (P<0.05) in the β-glucosidase activity over the 11

incubation period. Overall, the β-glucosidase activity of the tested strains increased during the incubation period (until 24 h) followed by a decline as fermentation progressed. The highest β-glucosidase activity of the strains occurred at 24 h except for L. plantarum GIM 1.35. Its highest β-glucosidase activity occurred at 12 h (1.20±0.047 u/ml), but this was not significantly different from that at 24 h (1.18±0.040 u/ml). By extending the incubation time, the β-glucosidase activity declined in a certain degree. The reason for this phenomenon might be ascribed to the weakened vitality and metabolic efficiency of the microbe in the later period of fermentation (Keppler & Humpf, 2005). The productivity of β-glucosidase depended on strain specificity and it varied apparently with intestinal beneficial bacterial growth cycles (Otieno, Ashton, & Shah, 2005). There was no difference among β-glucosidase activities of L. acidophilus GIM 1.83, B. animalis GIM 1.169 and L. delbrueckii subsp. Bulgaricus GIM 1.155, and their highest β-glucosidase activity all occurred at 24h (0.58±0.052 u/ml). This result was consistent with that for L. casei incubated in soymilk reported by Otieno et al. (2005). This is perhaps attributed to a different carbon source, which was the main factor affecting β-glucosidase activity (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009). Among all tested strains, L. plantarum GIM 1.35 and S. thermophiles GIM 1.321 had rather high β-glucosidase activities, 1.20 u/ml and 1.27 u/ml, respectively, 2.03-2.68 times of other three strains. In the literature, L. plantarum KFRI 00144 presented the highest β-glucosidase activity when compared with other lactobacilli and bifidobacteria strains (Pyo, Lee, & Lee, 2005); L. plantarum IFPL722 and L. casei strains showed 12

the highest cell-envelope associated β-glucosidase activity among nine tested strains (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009). Apart from the strains specificity, we predicted that strain viability also affected β-glucosidase activity. It was worth noting that the increase in β-glucosidase activity and the subsequent decline apparently corresponded to the strains growth cycle. These results were in agreement with others, who also demonstrated a direct correlation between β-glucosidase and the growth properties of bifidobacterium (Tsangalis, Ashton, McGill, & Shah, 2002). Keppler and Humpf (2005) even predicted β-glucosidase activity, expressed by the degradation of p-nitrophenol, could be a marker for the vitality and metabolic efficiency of the microflora. Fleschhut et al. (2006) also reported β-glucosidase activity changed along with the strain growth cycle. In our study, the exponential growth phase of most tested strains was between 12 and 24 h. Thus, it was easy to understand that peak enzyme activity occurred between 12 and 24 h among those strains.

3.3 Chemical stability and bacterial bioconversion of mulberry anthocyanins The chemical stability of mulberry anthocyanin under physiological conditions (37 oC, pH 6.0), was determined by incubating the mulberry anthocyanin without microbes. Though the glycoside molecule conferred stability to anthocyanin, anthocyanin degradation increased as pH, temperature, light, oxygen, and the number of hydroxyl group increased (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009). As can be seen from Fig. 3 (the control), anthocyanin 13

glycosides presented in mulberry underwent chemical degradation during incubation in MRS medium at 37 oC. From the initial concentration of about 80 mg L-1, around 19.42% were degraded within 48 h. Similarly, it was reported non-enzymatic deglycosylation of flavonoids existed in physiological conditions, even at the heavily acidic conditions of the stomach (Keppler & Humpf, 2005). To evaluate anthocyanin bioconversion by intestinal bacterial, preparations were incubated in batch culture fermenters. Mulberry anthocyanin biotransformation was observed in different levels among these test strains (Fig.3). Unexpectedly, compared with the control, biodegradation of mulberry anthocyanins in the presence of different probiotics was relatively laggard except for L. plantarum GIM 1.35 and S. thermophiles GIM 1.321 (Fig.3). A typical example could be found in B. animalis GIM 1.169 run, of which total residue anthocyanin kept a higher level than control until 24 h. A possible reason was that anthocyanins might interact with the protein presented in the broth, which somehow protected anthocyanins from degradation (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009). Once acidified methanol added, anthocyanins could be released from their linkage with proteins and consequently be quantified (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009; Fleschhut, Kratzer, Rechkemmer, & Kulling, 2006). Biodegradation of anthocyanins due to microorganism hydrolytic enzyme activity was obvious for L. plantarum GIM 1.35 and S. thermophiles GIM 1.321. Residual anthocyanins in groups of L. plantarum GIM 1.35 (44.59±1.81 mg L-1) and 14

S. thermophiles GIM 1.321 (43.92±1.14 mg L-1) were 65.98% and 65.04% of the control after 48 h incubation, respectively. Corresponding to our results, higher anthocyanin degradation efficiency was found in L. plantarum IFPL722 (66.4% residual

malvidin-3-glucoside)

than

B.

lactis

BB-12

(74.8%

residual

malvidin-3-glucoside) in 5 hours fermentation (Ávila, Hidalgo, Sánchez-Moreno, Pelaez, Requena, & de Pascual-Teresa, 2009). As previously stated, dramatically high β-glucosidase activity for L. plantarum GIM 1.35 and S. thermophiles GIM 1.321 was detected (Fig. 2) which might translated into stronger deglycosylation of cyanidin-3-glucoside or cyanidin-3-rutinoside. This could be also confirmed from the concentration changes of the main anthocyanins (Fig.4). L. plantarum GIM 1.35 exhibited the highest cyanidin-3-glucoside degradation efficiency while S. thermophiles GIM 1.321 exhibited the highest cyanidin-3-rutinoside degradation efficiency. After fermentation, nearly half of the cyanidin-3-glucoside and over 60% of cyanidin-3-rutinoside were degraded by L. plantarum GIM 1.35 and S. thermophiles GIM 1.321. It is worth noting that although β-glucosidase activity for S. thermophiles GIM 1.321 was lower than L. plantarum GIM 1.35 after 24 h, their total anthocyanin content changes were similar. This is perhaps attributed to cell lysis effects, which could release intracellular enzymes leading to anthocyanin degradation as opposed to the activity of intact bacterial cells (Ávila et al., 2009).

3.4 Biotransformation products of mulberry anthocyanins Studies have demonstrated that health effects of anthocyanins were not only due to anthocyanins themselves, but also to their metabolites produced by the gut 15

microflora (Hidalgo, et al., 2012). Therefore, the transformation of anthocyanin glycosides and their main metabolites were detected. All supernatants studied in the current study exhibited the ability to convert anthocyanin in mulberry concentrate (Fig.5). Since cyanidin-3-glucoside and cyanidin-3-rutinoside were the main anthocyanin glycosides detected in this system (Fig.S1), phenolic acid could have originated from syringic enzymatic demethylation of C3 of the B-ring (Fleschhut, Kratzer, Rechkemmer, & Kulling, 2006). The effect of bacterial metabolism resulted in the rupture of glycosidic linkages and cleavage of the anthocyanin heterocycle ring. The increment of peaks area could have resulted from anthocyanin degradation due to chemical or bacterial enzymatic degradation (Fig.S2). Previous reports demonstrated that protocatechuic acid and ferulic acid could be formed by spontaneous degradation from cyanidin, a degradation product of anthocyanins formed by B/C ring cleavage of cyanidin-3-O-glucoside (Seeram, Bourquin, & Nair, 2001). Ávila et al (2009) reported gallic acid, homogentisic acid, syringic acid, p-coumaric acid and sinapic acid as the main metabolites for malvidin-3-glucoside degradation. In this study, phenolic compounds, chlorogenic acid, cryptochlorogenic acid, caffeic acid and ferulic acid were detected as the main metabolites when mulberry anthocyanins were fermented with intestinal probiotics (Fig.S2). Without inoculation, all phenolic acids concentrations raised slowly during the whole period and the final concentrations were 10.23±1.15 mg L-1 (chlorogenic acid), 5.37±1.05 mg L-1 (cryptochlorogenic acid), 5.12±0.52 mg L-1 (caffeic acid) and 6.12±0.85 mg L-1 (ferulic acid), respectively (Fig.5). This result was not indicative of 16

anthocyanin biotransformation by strains, but rather of instability and chemical degradation. Protocatechuic acid and cyanidin aglycones have been demonstrated as the major metabolites for cyanidin-3-rutinoside (Aura, et al., 2005; Hassimotto, Genovese, & Lajolo, 2008), while limited studies have reported the chemical degradation metabolites of mulberry anthocyanins. Chemical breakdown of mulberry anthocyanins led mainly to the formation of chlorogenic acid, which was reported as having several beneficial biological properties including antibacterial, antiphlogistic, antiviral, mutant resistance, and even inhibitory effects on carcinogenesis in large intestine, liver, and tongue (Bagdas, Etoz, Gul, Ziyanok, Inan, Turacozen, et al., 2015; Hao, Gao, Liu, He, Tang, & Guo, 2016). Cryptochlorogenic acid, another main metabolite in the mulberry anthocyanin degradation, was also reported as a major bioactive components responsible for Reduning therapeutic (Wang, Wen, Zheng, Zhao, Fu, Wang, et al., 2015). For all runs, concentrations of those metabolites increased throughout the time period concomitant with anthocyanin degradation. When the strains reached the stationary phase, accumulation of the phenolic acids slowed down except for caffeic acid. This was perhaps because enzymes in intestinal bacteria responsible for anthocyanin degradation needs to be induced by anthocyanins (Knockaert, Raes, Wille, Struijs, & Van Camp, 2012). Ferulic acid content in the control was even higher than B. animalis GIM 1.169 and L. acidophilus GIM 1.83 runs. The reason for this phenomenon might be ascribed to consumption of phenolic acids by strains. The metabolism of phenolic acids, ferulic acid, during growth of Lactobacillus plantarum and Lactobacillus collinoides have also been reported by 17

others (Knockaert, Raes, Wille, Struijs, & Van Camp, 2012). Overall, in corresponding to the β-glucosidase activity variations, L. plantarum GIM 1.35 and S. thermophiles GIM 1.321 obtained higher polyphenols yields than that of other three strains. Within 48 h fermentation, L. plantarum GIM 1.35 achieved the highest concentration ferulic acid (8.18±0.37 mg L-1), while S. thermophiles GIM 1.321 yielded the highest amount of chlorogenic acid (26.24±0.54 mg L-1) and caffeic acid (6.33±0.36 mg L-1). For their higher chemical and microbial stability, these phenolic acids and/or other not yet identified anthocyanin metabolites might be partly responsible for mulberry antioxidant activities and other physiological effect.

Conclusion Mulberry anthocyanin bioconversion by intestinal beneficial bacteria was investigated in this study. Mulberry anthocyanins were degraded 31.24-46.17% by five intestinal beneficial bacteria and bioavailable metabolites, protocatechuic acid, chlorogenic acid and ferulic acid, were produced through bacterial enzymatic actions. The most important enzyme related to this biotransformation was β-glucosidase which is secreted by the tested intestinal beneficial bacteria. The productivity of this enzyme varied apparently with intestinal beneficial bacterial growth cycles and the highest β-glucosidase activity occurred during the late exponential phase (18-24 h). S. thermophiles GIM 1.321 and L. plantarum GIM 1.35 exhibited the highest β-glucosidase production potential while B. animalis GIM 1.169 exhibited the lowest. Non-enzymatic deglycosylation of mulberry anthocyanins was also found and about 19.4% of the anthocyanins were degraded within 48 h. These results suggest that 18

mulberry anthocyanin bioavailability could be improved by prior degradation to obtain more phenolic acids in situ in health probiotic foods, although further research should be carried out to understand the pharmacological roles of mulberry anthocyanin metabolites.

Acknowledgements The authors gratefully acknowledge the financial support of Guangzhou Science and Technology Program key projects [grant No. 2014J2200068]; Guangdong Province

Science

and

Technology Program

[grant

No. 2013B050800018;

2012B091100468; grant No.2014B040404059; grant No.2013B090600084]. Authors also acknowledge David Hopkins for the assistance on manuscript modification.

References Araceli, C. O., Lourdes, P. H., Ma, E. P.-H., Jose, A. R., & Carlos, A. G. V. (2009). Chemical studies of anthocyanins: A review. Food Chemistry, 113(4), 859-871. Aura, A. M. (2008). Microbial metabolism of dietary phenolic compounds in the colon. Phytochemistry Reviews, 7(3), 407-429. Aura, A. M., Pilar, M. L., Karen Anne, O. L., Gary, W., Kirsi-Marja, O. C., Kaisa, P., & Celestino, S. B. (2005). In vitro metabolism of anthocyanins by human gut microflora. European Journal of Nutrition, 44(3), 133-142. Ávila, M., Hidalgo, M., Sánchez-Moreno, C., Pelaez, C., Requena, T., & de Pascual-Teresa, S. (2009). Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus. 19

Food Research International, 42(10), 1453-1461. Bagdas, D., Etoz, B. C., Gul, Z., Ziyanok, S., Inan, S., Turacozen, O., Gul, N. Y., Topal, A., Cinkilic, N., & Tas, S. (2015). In vivo systemic chlorogenic acid therapy under diabetic conditions: Wound healing effects and cytotoxicity/genotoxicity profile. Food and Chemical Toxicology, 81, 54-61. Bridle, P., & Timberlake, C. (1997). Anthocyanins as natural food colours—selected aspects. Food Chemistry, 58(1), 103-109. Bub, A., Watzl, B., Heeb, D., Rechkemmer, G., & Briviba, K. (2001). Malvidin-3-glucoside bioavailability in humans after ingestion of red wine, dealcoholized red wine and red grape juice. European Journal of Nutrition, 40(3), 113-120. Fleschhut, J., Kratzer, F., Rechkemmer, G., & Kulling, S. E. (2006). Stability and biotransformation of various dietary anthocyanins in vitro. European Journal of Nutrition, 45(1), 7-18. Flores, G., Ruiz del Castillo, M. L., Costabile, A., Klee, A., Bigetti Guergoletto, K., & Gibson, G. R. (2015). In vitro fermentation of anthocyanins encapsulated with cyclodextrins: Release, metabolism and influence on gut microbiota growth. Journal of Functional Foods, 16(0), 50-57. Forester, S. C., & Waterhouse, A. L. (2008). Identification of Cabernet Sauvignon anthocyanin gut microflora metabolites. Journal of Agricultural and Food Chemistry, 56(19), 9299-9304. Ghasemzadeh, A., Jaafar, H. Z. E., & Karimi, E. (2012). Involvement of Salicylic Acid on Antioxidant and Anticancer Properties, Anthocyanin Production and Chalcone Synthase Activity in Ginger (Zingiber officinale Roscoe) Varieties. International Journal of 20

Molecular Sciences, 13(11), 14828-14844. Hao, Y., Gao, R., Liu, D., He, G., Tang, Y., & Guo, Z. (2016). Selective extraction and determination of chlorogenic acid in fruit juices using hydrophilic magnetic imprinted nanoparticles. Food Chemistry, 200, 215-222. Hassimotto, N. M. A., Genovese, M. I., & Lajolo, F. M. (2008). Absorption and metabolism of cyanidin-3-glucoside and cyanidin-3-rutinoside extracted from wild mulberry (Morus nigra L.) in rats. Nutrition Research, 28(3), 198-207. Hidalgo, M., Martin-Santamaria, S., Recio, I., Sanchez-Moreno, C., de Pascual-Teresa, B., Rimbach, G., & de Pascual-Teresa, S. (2012). Potential anti-inflammatory, anti-adhesive, anti/estrogenic, and angiotensin-converting enzyme inhibitory activities of anthocyanins and their gut metabolites. Genes and Nutrition, 7(2), 295-306. Hosoda, M., Hashimoto, H., He, F., Morita, H., & Hosono, A. (1996). Effect of administration of milk fermented with Lactobacillus acidophilus LA-2 on fecal mutagenicity and microflora in the human intestine. Journal of dairy science, 79(5), 745-749. Jeon, K. S., Ji, G. E., & Hwang, I. K. (2002). Assay of β-glucosidase activity of bifidobacteria and the hydrolysis of isoflavone glycosides by Bifidobacterium sp. Int-57 in soymilk fermentation. Journal of microbiology and biotechnology, 12(1), 8-13. Keppler, K., & Humpf, H.-U. (2005). Metabolism of anthocyanins and their phenolic degradation products by the intestinal microflora. Bioorganic & Medicinal Chemistry, 13(17), 5195-5205. Knockaert, D., Raes, K., Wille, C., Struijs, K., & Van Camp, J. (2012). Metabolism of ferulic acid during growth of Lactobacillus plantarum and Lactobacillus collinoides. Journal of the 21

Science of Food and Agriculture, 92(11), 2291-2296. Liang, L., Wu, X., Zhao, T., Zhao, J., Li, F., Zou, Y., Mao, G., & Yang, L. (2012). In vitro bioaccessibility and antioxidant activity of anthocyanins from mulberry (Morus atropurpurea Roxb.) following simulated gastro-intestinal digestion. Food Research International, 46(1), 76-82. Liu, X., Xiao, G., Chen, W., Xu, Y., & Wu, J. (2004). Quantification and purification of mulberry anthocyanins with macroporous resins. BioMed Research International, 2004(5), 326-331. Luo, Q., Zhang, J., Yan, L., Tang, Y., Ding, X., Yang, Z., & Sun, Q. (2011). Composition and Antioxidant Activity of Water-Soluble Polysaccharides from Tuber indicum. Journal of Medicinal Food, 14(12), 1609-1616. Manach, C., Williamson, G., Morand, C., Scalbert, A., & Remesy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. The American journal of clinical nutrition, 81(1 Suppl), 230S-242S. Markham, K., Geiger, H., & Harborne, J. (1994). The Flavonoids: Advances in research since 1986. Chap, 10, 441-493. Meurman, J., Antila, H., Korhonen, A., & Salminen, S. (1995). Effect of Lactobacillus rhamnosus strain GG (ATCC 53103) on the growth of Streptococcus sobrinus in vitro. European journal of oral sciences, 103(4), 253-258. Netzel, M., Strass, G., Janssen, M., Bitsch, I., & Bitsch, R. (2001). Bioactive anthocyanins detected in human urine after ingestion of blackcurrant juice. Journal of environmental pathology, toxicology and oncology, 20(2). 22

Otieno, D., Ashton, J., & Shah, N. E. (2005). Stability of β‐glucosidase Activity Produced by Bifidobacterium and Lactobacillus spp. in Fermented Soymilk During Processing and Storage. Journal of food science, 70(4), M236-M241. Pavlovic, A. V., Dabic, D. C., Momirovic, N. M., Dojcinovic, B. P., Milojkovic-Opsenica, D. M., Tesic, Z. L., & Natic, M. M. (2013). Chemical Composition of Two Different Extracts of Berries Harvested in Serbia. Journal of Agricultural and Food Chemistry, 61(17), 4188-4194. Pyo, Y. H., Lee, T. C., & Lee, Y. C. (2005). Enrichment of bioactive isoflavones in soymilk fermented

with

β-glucosidase-producing

lactic

acid

bacteria.

Food

Research

International, 38(5), 551-559. Rolfe, R. D. (2000). The role of probiotic cultures in the control of gastrointestinal health. The Journal of Nutrition, 130(2), 396S-402S. Seeram, N. P., Bourquin, L. D., & Nair, M. G. (2001). Degradation products of cyanidin glycosides from tart cherries and their bioactivities. Journal of Agricultural and Food Chemistry, 49(10), 4924-4929. Shi, Z. U., Bassa, I. A., Gabriel, S. L., & Francis, F. J. (1992). Anthocyanin Pigments of Sweet Potatoes–Ipomoea batatas. Journal of food science, 57(3), 755-757. Stead, D. (1993). The effect of hydroxycinnamic acids on the growth of wine‐spoilage lactic acid bacteria. Journal of applied bacteriology, 75(2), 135-141. Szwajgier, D., & Jakubczyk, A. (2010). Biotransformation of ferulic acid by Lactobacillus acidophilus K1 and selected Bifidobacterium strains. Acta Scientiarum Polonorum. Technologia Alimentaria, 9(1), 45-59. 23

Tobias, J. (1968). Dairy technology in a food science department. Journal of dairy science, 51(2), 255-259. Tsangalis, D., Ashton, J., McGill, A., & Shah, N. (2002). Enzymic Transformation of Isoflavone Phytoestrogens in Soymilk by β‐Glucosidase‐Producing Bifidobacteria. Journal of food science, 67(8), 3104-3113. Wang, Y., Wen, J., Zheng, W., Zhao, L., Fu, X., Wang, Z., Xiong, Z., Li, F., & Xiao, W. (2015). Simultaneous determination of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and geniposide in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study after administration of Reduning injection. Biomedical Chromatography, 29(1), 68-74. Yan, J. J., Jung, J. S., Hong, Y. J., Moon, Y. S., Suh, H. W., Kim, Y. H., Yun-Choi, H. S., & Song, D. K. (2004). Protective Effect of Protocatechuic Acid Isopropyl Ester against Murine Models of Sepsis: Inhibition of TNF-α. ALPHA. and Nitric Oxide Production and Augmentation of IL-10. Biological and Pharmaceutical Bulletin, 27(12), 2024-2027.

24




Mulberry anthocyanin bioconversion properties of five different intestinal beneficial bacteria were investigated for the first time.




S. thermophiles GIM 1.83 and L. plantarum GIM 1.35 showed the highest Mulberry anthocyanin bioconversion potential, while B. animalis GIM 1.169 was the lowest.


Bioavailable metabolites, such as protocatechuic acid, chlorogenic acid, caffeic acid, and ferulic acid, were detected in Mulberry anthocyanin bioconversion.

25