Bacterial Cinnamoyl Esterase Activity Screening for the Production of a Novel Functional Food Product

Lactobacillus helveticus MIMLh5 was selected for its strongcinnamoyl esterase activity on chlorogenic acid and employedfor the preparation of a food product containing a high concentrationof free caffeic acid. The novel food product was demonstratedto display high total antioxidant power and potential probioticproperties.

INTRODUCTION

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INTRODUCTION

Hydroxycinnamic acids such as caffeic, ferulic, sinapic, andp-coumaric acids are found in almost every plant (11) and representa major class of phenolic compounds. The most abundant hydroxycinnamicacid is caffeic acid, which is contained in foods mainly asester with quinic acid, a form called chlorogenic acid (5-caffeoylquinicacid) (Fig. 1).

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FIG. 1. Structures of chlorogenic acid and caffeic acid. The arrow indicates the esterase bond hydrolyzed by cinnamoyl esterase.

Caffeic acid and chlorogenic acid have been demonstrated previouslyto possess remarkably high levels of antioxidant activity invitro (8, 12, 13, 18, 25), and they have been associated withseveral health-promoting effects in vivo (17, 27-30, 33).

The biological properties of the hydroxycinnamic acids dependon their absorption in the gastrointestinal tract. It has beendemonstrated previously that the absorption of caffeic acidis drastically reduced when this molecule is ingested as chlorogenicacid (10, 14, 20). The majority of chlorogenic acid, in fact,reaches the large intestine, where it is quickly hydrolyzedby bacteria with cinnamoyl esterase activity and extensivelyfurther degraded by the microbiota (10). In contrast, free caffeicacid is well absorbed in the stomach and in the small intestine(20, 23), and its intake is associated with a higher concentrationof intact caffeic acid and its tissular metabolites in plasmaand a higher level of urinary excretion of these substancesthan those resulting from the intake of chlorogenic acid (10).

Cinnamoyl esterases have commonly been found in rumen and soilsaprophytic microorganisms (6, 7, 16) and also in bacteria fromhuman and animal intestinal microbiota (5, 32). However, veryfew reports describing cinnamoyl esterases of food-associatedbacteria are available (6).

In this study, 100 food and human intestinal bacterial strains(Table 1) were screened for the production of cinnamoyl esteraseactivity by thin-layer chromatography on silica gel 60 F254plates (Merck, Milan, Italy). Bacterial strains were grown in1 ml of liquid medium for 72 h in the presence of a 0.5-mg/mlconcentration of the reference substrate, ethyl ferulate orchlorogenic acid (Sigma, St. Louis, MO). Only 12 strains gaveundoubtedly positive results on both substrates, and these strainsbelonged to the species Lactobacillus helveticus, L. acidophilus,and L. fermentum (Table 2).

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TABLE 1. Bacterial strains screened for cinnamoyl esterase activity

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TABLE 2. Enzymatic activity of the 24 bacterial strains testing positive for the presence of cinnamoyl esterase

The percentages of chlorogenic acid hydrolyzed by the positivebacterial strains were determined by high-performance liquidchromatography (HPLC) analysis. The HPLC apparatus consistedof an Alliance 2695 pump system (Waters, Milford, MA) equippedwith a Waters 996 diode array detector. A Hypersil AA octyldecylsilane column (2.1-mm internal diameter by 250-mm length; filmthickness, 5 µm; pore size, 120 Å) from AgilentTechnologies (Santa Clara, CA) kept at 30°C was used. Elutingsolvents were water containing 4% (vol/vol) formic acid (Sigma)and methanol (Merck, Darmstadt, Germany). The linear elutiongradient (200 µl/min) expressed as the methanol proportionwas as follows: 0 min, 5%; 0 to 5 min, 5%; 5 to 35 min, 5 to20%; 35 to 36 min, 20 to 100%; 36 to 38 min, 100%; 38 to 39min, 5% (run-to-run time, 50 min). UV spectra of chromatographicpeaks were acquired by scanning the range from 210 to 400 nm(resolution, 1.2 nm).

Two strains belonging to the L. helveticus species, named MIMLh5and SIM7, apparently hydrolyzed all the chlorogenic acid usedin the experiments (Table 2). In particular, the L. helveticusstrain MIMLh5, isolated from an Italian cheese natural wheystarter culture, showed good growth in milk and in apple juice,and therefore, it was chosen as a starter for the preparationof the food product.

Development of fermented food product enriched with caffeic acid.

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Development of fermented food...

The main intention of this work was the preparation of a foodproduct containing a significant concentration of free caffeicacid, obtained from the chlorogenic acid naturally present inthe food through the esterase activity of a food-grade bacterialstarter. We selected the ingredients of the food product withthe purposes of obtaining an alimentary formulation naturallyrich in chlorogenic acid and creating the conditions suitablefor the growth of the microbial starter. More specifically (Fig.2), we chose Renetta cultivar apples because they revealed thehighest content of phenolic compounds among the best-known applecultivars (31), and we included a green tea infusion becauseof the high concentration of antioxidants, and particularlychlorogenic acid, in green tea (22); ascorbic acid preventedthe browning of the apple pulp during production, and the presenceof skim milk promoted the growth of the bacterial starter. Nevertheless,we verified good proliferation of the bacterial starter evenin the absence of skim milk and in the presence of soy milk.

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FIG. 2. Flow sheet depicting the preparation of the fermented food product.

After fermentation, the food product had the texture of yogurtand a mixed flavor of yogurt and apple. The taste was fresh,and the flavors of apple and green tea were dominant. The homolacticfermentation by the microbial starter reduced the pH from about7 to 3.6.

At the end of the fermentation process, the final biomass was8.6 x 10⁸ cells/ml of the food product. Experiments with propidiumiodide (1-µg/ml final concentration; Sigma) and countson MRS agar plates supplemented with 0.05% cysteine hydrochlorideshowed a log decrease in viable cells not higher than 0.8 after3 weeks of storage at 4°C.

The quantitation of caffeic and chlorogenic acids in unfermentedand fermented food products was carried out by HPLC at 320 nmas described above. For the extraction of hydroxycinnamic acids,the pH of the food product was brought to 1.5 with HCl and 1g of the acidified sample was incubated twice with stirringat 4°C for 1 h in 5 ml of methanol and 0.1% ascorbic acid.Standard solutions of caffeic and chlorogenic acids in methanolat a concentration of 1,000 mg/liter were prepared and graduallydiluted. Both spiked and unknown samples were run in duplicate,and three independent determinations were done. The assignmentof chromatographic peaks to caffeic and chlorogenic acids wasfurther confirmed by UV spectra and HPLC-electrospray ionization-massspectrometry experiments. Particularly, the recognition of hydroxycinnamicacids was carried out by different means: (i) selected-ion-monitoringscanning (peak width, 1.0 Da) for specific monoprotonated ions[(M + H⁺)¹⁺; m/z, 181.2 and 355.3 for caffeic and chlorogenicacids, respectively] and (ii) a full scan at m/z 100 to 400.The mass accuracy was ensured by calibration with a mixtureof caffeine and reserpine and the tripeptide PFK (in a 1:1 methanol-watersolution with 0.1% acetic acid) infused separately. After 70h of fermentation, chromatographic peaks with retention timescorresponding to those of standard chlorogenic (23.0 min) andcaffeic (19.8 min) acids were present in the HPLC pattern ofthe fermented food product (Fig. 3). The bacterial starter convertedmost of the chlorogenic acid into caffeic acid, the concentrationof which increased from 6.4 ± 3.9 to 149 ± 11mg/liter during the fermentation process (results are shownas means ± standard deviations; n = 3).

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FIG. 3. HPLC chromatograms of the methanolic extracts of the food product before (A) and after (B) the fermentation with L. helveticus MIMLh5. C, standard solution of caffeic and chlorogenic acids.

The amount of caffeic acid ingested in consuming, for instance,100 ml of the food product described here may significantlyincrease the total daily amount of absorbed phenolic compounds(24) and may have a significant biological effect on the consumer(4, 19, 26). Indeed, studies of rats and humans have shown that,different from chlorogenic acid, caffeic acid is efficientlyabsorbed through the upper part of the gastrointestinal tractand is found in the body either intact or in glucuronidated,sulfated, and O-methylated forms (ferulic and isoferulic acids),increasing the total antioxidant status of plasma (19, 20, 23).

Other potentially functional features.

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Other potentially functional...

We verified the total antioxidant capacity by evaluating theability of the food product to reduce Fe³⁺ ions into Fe²⁺ ionsunder acidic conditions as demonstrated by assaying the ferricreducing ability of plasma (see the supplemental material fordetails on the experimental protocol), a method already successfullyapplied to a variety of food products (3, 21). The total antioxidantcapacity of our fermented product proved to be much higher thanthose of the other fermented dairy products tested and alsohigher than those of beverages with high concentrations of antioxidantcompounds, such as red wine and black tea (Fig. 4), suggestingthat the fermented food described herein can be considered anantioxidant alimentary product. The antioxidant power of thefermented product was not significantly different from thatof the unfermented food. This result indicates that the activityof the bacterial starter did not alter the reducing potentialof the food or eventually counteracted its loss.

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FIG. 4. Antioxidant activities of 10 different food products as determined by assaying the ferric reducing ability of plasma. RAU, relative antioxidant units; FFP, functional food product developed in this work; nf-FP, nonfermented FFP; ACE juice, commercial orange and carrot juice enriched with vitamins A, C, and E; Actimel, probiotic fermented milk from Danone. Vertical bars represent standard deviations. Unpaired Student's t test revealed a statistically significant difference between FFP and nf-FP and either red wine or green tea (P < 0.05).

L. helveticus MIMLh5 belongs to a species that is generallyrecognized as safe and has been often associated with health-promotingeffects (1, 15). We studied its ability to resist gastrointestinaltransit and to bind to intestinal epithelial cells in vitro(data not shown; see the supplemental material for details onthe experimental protocols). Experiments with simulated gastricjuices at pH 2 and pH 3 showed that L. helveticus MIMLh5 cansurvive for 3 h, very similar to the control strain L. rhamnosusGG, which is the most studied probiotic and is known to survivegastrointestinal transit (9). We also observed that MIMLh5 cansurvive very well in the presence of 0.3% oxgall and can growin MRS liquid medium supplemented with 0.05% cysteine hydrochlorideand 0.2% oxgall. Finally, we found that L. helveticus MIMLh5can adhere to cells of the Caco-2 human epithelial cell line,very similar to the control strain L. rhamnosus GG, which isknown to be able to transiently colonize the human intestinaltract (2) (data not shown).

In conclusion, the alimentary product prepared and characterizedin this work is a novel functional food exhibiting high antioxidantpower and presenting probiotic potential. Furthermore, the noveltyof the present work resides in the use of a bacterial probioticstarter, selected for its high level of cinnamoyl esterase activity,for the production of a significant concentration of free caffeicacid during the fermentation of a food matrix rich in antioxidants(S. Guglielmetti and D. Mora, 27 April 2007, European patentapplication EP 07008593.1). The promising potential health-promotingeffects of this food product need to be proven by upcoming studiesof human nutrition and physiology.

ACKNOWLEDGMENTS

This work was supported by a grant from the Ministry of theUniversity and Technological and Scientific Research (First2005).

We are grateful to Daniele Ramponi for his invaluable technicalassistance.

FOOTNOTES

* Corresponding author. Mailing address: Department of Food Science and Microbiology, Industrial Microbiology Section, University of Milan, Via Celoria 2, 20133 Milan, Italy. Phone: 39 02 503 19136. Fax: 39 02 503 19292. E-mail: simone.guglielmetti{at}unimi.it

Published ahead of print on 28 December 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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