The aim of this study was to investigate the antimicrobial activity of two Yucca filamentosa L.
extracts produced by hydro-alcoholic extraction under different operational conditions. The extracts were concentrated and de-alcoholised, and were added to media to test their effects on target microorganisms. Both extracts displayed equivalent specific growth inhibitory activity against
Saccharomyces cerevisiae. No other antimicrobial activity was found. This anti-fermentative activity may open the doors to new possibilities for using Yucca filamentosa L. extracts in the food industry. These findings are of particular interest, since yucca extracts are already classified as
being generally recognized as safe (GRAS)

F. Tateo

G. Guglielmi

M. Bononi

P. Rocchi

The aim of this study was to investigate the antimicrobial activity of two Yucca filamentosa L.
extracts produced by hydro-alcoholic extraction under different operational conditions. The extracts were concentrated and de-alcoholised, and were added to media to test their effects on target microorganisms. Both extracts displayed equivalent specific growth inhibitory activity against
Saccharomyces cerevisiae. No other antimicrobial activity was found. This anti-fermentative activity may open the doors to new possibilities for using Yucca filamentosa L. extracts in the food industry. These findings are of particular interest, since yucca extracts are already classified as
being generally recognized as safe (GRAS)

AIR Universita degli studi di Milano

ISSN 1120-1770ITALIAN JOURNALOFFOOD SCIENCERivista  italianadi scienza degli alimentiVolume XXVNumber 12013CHIRIOTTI          EDITORICOP IJFS 25 anniversary_Layout 1  05/03/13  18:36  Pagina 1 u r 22013short communication238 Ital. J. Food Sci., vol. 25 - 2013- Keywords: Yucca filamentosa L., antimicrobial activity, yucca extracts -FIRST DATA ON THE ANTIMICROBIAL ACTIVITYOF YUCCA FILAMENTOSA L. BARK EXTRACTSM. BONONI, G. GUGLIELMI1, P. ROCCHI1 and F. TATEO*Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy1Rocchi dr. Eugenio s.r.l., Via J.F. Kennedy 3, 40053 Bazzano, BO, Italy*Corresponding author: Tel. +39 02 50316540, Fax +39 02 50316539,email: fernando.tateo@ .itAbStrActthe aim of this study was to investigate the antimicrobial activity of two Yucca filamentosa L. extracts produced by hydro-alcoholic extraction under different operational conditions. the ex-tracts were concentrated and de-alcoholised, and were added to media to test their effects on tar-get microorganisms. both extracts displayed equivalent specific growth inhibitory activity against Saccharomyces cerevisiae. No other antimicrobial activity was found. this anti-fermentative ac-tivity may open the doors to new possibilities for using Yucca filamentosa L. extracts in the food industry. these findings are of particular interest, since yucca extracts are already classified as being generally recognized as safe (GrAS). Ital. J. Food Sci., vol. 25 - 2013 239INtrODUctIONthe various Yucca species (fam. Asparagaceae, sub-fam. Agavoideae) are tropical or sub-tropi-cal plants with a tree-like growth habit. they are found in arid or semi-arid South-Western regions of the US and Mexico (PELLMYr, 2003). In tra-ditional medicine of the Native Americans, yuc-ca-derived extracts were used against a variety of diseases, including arthritis and rheumatism (MUKHErJEE et al., 2001); these extracts have GrAS (generally recognized as safe) status and are therefore accepted by the US FDA for use in humans. Yucca powder and sap are derived from the logs of the plant; such extracts can be pro-duced by mechanical squeezing and subsequent evaporation of the sap, and are widely used in food, cosmetics, and pharmaceutics (MAttIA and cOLUZZI, 2005). Yucca powder and extracts are also used as animal feed additives due to their beneficial anti-inflammatory effects (WENZIG et al., 2008), which have been linked to their high content of phenolic compounds.Many studies have also examined the sapo-nin-rich extracts of different Yucca species for antibacterial activity (cHEEKE et al., 2006; FAVEL et al., 2005; KILLEN et al., 1998; KOWALcZYK et al., 2011; MILGAtE and rObErtS, 1995; MIYA-KOSHI et al., 2000; MONtOrO et al., 2008; PIA-cENtE et al., 2005; WENZIG et al., 2008). Sap-onins are glycoside compounds of a fat-soluble nucleus (aglycone) that can be either a triterpe-noid (c30), as in soybean, alfa-alfa, quillaja, and guar (HASSAN et al., 2010); or an alkaloid steroid (c27), as in yucca, tomato, and oats. One or more side chains or water-soluble sugars (glycone) are attached through ester linkages to the aglycone nucleus at different carbon sites (KANEDA et al., 1987). Saponins have shown haemolytic (KHALIL and EI-ADAWY, 1994) and antibacterial activities (SEN et al., 1998), but not all saponins have the same biological activities (HASSAN et al., 2010, KANEDA et al., 1987). It has been demonstrated that yucca bark accumulates polar bidesmosid-ic saponins; the bark powder samples are domi-nated by high polarity, while steroidal glycosides with mid- and short-length saccharide chains are predominant in the steam. A large number of papers have determined that Yucca schidigera contains steroidal sapo-nins with anti-yeast activity, and therefore such extracts have been investigated for use as new agents to prevent food deterioration (MIYAKOSHI et al., 2000). Our preliminary experiments show similar properties exhibited by root extracts of Yucca filamentosa L. from the USA, also studied previously addressing the importance of chem-istry and geographic variation of floral scent (SVENSSON et al., 2005). In the present study, we tested the efficiencies of producing Yucca fil-amentosa L. extract using different extraction solvents, and then tested the antimicrobial ac-tivities of these extracts vs. several target mi-croorganisms. We found evidence that the ex-tracts had considerable microbicidal activity ex-clusively against Saccharomyces cerevisiae and not against other target microorganisms.MAtErIAL AND MEtHODSExtract production for microbiological testingDuring the extraction, and particularly during separation of the solid waste, we attempted to avoid introducing technologically differentiated parameters for the various extracts by adopting the simple spontaneous drainage, without affect-ing by pressure the release of active principles from solid. the dry vegetable material of Yucca filamentosa L. was cut into pieces with an aver-age diameter of 10 mm. Preliminary extractions were performed under varying conditions, in or-der to identify the parameters that produced the best results in terms of percentage of the total soluble solids in the extracts. Extractions were performed by seven days of infusion of dry materials with solvent in a solid:liquid ratio of 1:6.7; the extraction mixture was shaken three times per day. three different solvents were used: (A) water, (b) water/95% eth-anol (50:50 v/v), and (c) 95% ethanol. In theory, derivation using different types of solvents could result in the extracts having different composi-tions of the active substances.Extraction efficiencies were measured (table 1). Solvent b was determined to produce the best ex-tractive yield. A new extract was produced using solvent b, this time cutting the dry yucca to piec-es with an average diameter of 1-2 mm. this ex-traction product was concentrated in a rotovapor (2:1); the obtained product was named extract b and contained a concentration of total soluble sol-ids equal to 14.0 g 100 mL-1. We also combined the original extracts produced with solvents A, b, and c, in a ratio of 1:1:1. this mixture was concentrated in a rotovapor (4:1) to produce an extract with total soluble solids concentration of 14.2 g 100 mL-1, similar to that of the concentrat-ed extract b. both concentrated products had a residual ethanol content of no more than 0.01%. the concentrated extracts b and Abc were next tested for their activity on microorganisms. Microbiological verificationsFor testing the antimicrobial activity of the yucca extracts, the following species were used as target microorganisms: Aspergillus brasil-iensis (NcPF 2275/Atcc 16404), Escherichia coli (Nctc 12241/Atcc 25922), Staphylococ-cus aureus (Nctc 12981/Atcc 25923), Lis-teria monocytogenes (Nctc 11994), Bacillus ce-reus (Nctc 7464/Atcc 10876), and Saccha-romyces Cerevisiae (NcPF 3178). these strains 240 Ital. J. Food Sci., vol. 25 - 2013table 1 - Preliminary results of testing three different extraction solvents for Yucca filamentosa L. Yucca (g) 95% EtOH H20 (mL) Extract Total Soluble  (mL)  (mL)  Solids (g 100 mL-1)Solvent A 888 6,000 --- 4,330 0.56Solvent B 878 3,000 3,000 3,800 5.32Solvent C 880 --- 6,000 2,400 3.58table 2 - results concerning growth values as cfu mL-1 derived from sowing stock suspension on undiluted media and media modified with two yucca extracts (Abc and b). counts on tryptone soy agar (t.s.a.) and Sabouraud dextrose agar (s.a.) me-dia are reported. the values are the average of four replicas, calculated according to ISO 8199:2005 Standard (8.4.2); stand-ard deviation (S) and p values are reported for the significant growth reduction of S. cerevisiae.  Medium cfu mL-1 95% Confidence Interval Log 95% Confidence Interval   (cfu mL-1) cfu mL-1 (Log cfu mL-1)Bacillus t.s.a. 1.456 × 106 1.451 × 106 1.461 × 106 6.163 6.162 6.165cereus t.s.a. + ABC 1.475 × 106 1.470 × 106 1.480 × 106 6.169 6.167 6.170 t.s.a. + B 1.341 × 106 1.337 × 106 1.346 × 106 6.127 6.126 6.129Escherichia t.s.a. 23.249 × 106 23.159 × 106 23.339 × 106 7.366 7.365 7.368coli t.s.a. + ABC 34.424 × 106 34.290 × 106 34.558 × 106 7.537 7.535 7.539 t.s.a. + B 24.324 × 106 24.236 × 106 24.411 × 106 7.386 7.384 7.388Staphylococcus t.s.a. 4.236 × 106 4.221 × 106 4.250 × 106 6.627 6.625 6.628aureus t.s.a. + ABC 4.252 × 106 4.237 × 106 4.268 × 106 6.629 6.627 6.630 t.s.a. + B 5.026 × 106 5.005 × 106 5.047 × 106 6.701 6.699 6.703Listeria  t.s.a.  26.424 × 106 26.322 × 106 26.526 × 106 7.422 7.420 7.424monocytogenes t.s.a. + ABC 20.999 × 106 20.916 × 106 21.082 × 106 7.322 7.320 7.324 t.s.a. + B 28.126 × 106 28.013 × 106 28.238 × 106 7.449 7.447 7.451Aspergillus s.a. 31.500 × 103 30.706 × 103 32.294 × 103 4.498 4.487 4.509brasiliensis s.a. + ABC 29.500 × 103 28.732 × 103 30.268 × 103 4.470 4.458 4.481 s.a. + B 33.500 × 103 32.681 × 103 34.319 × 103 4.525 4.514 4.536Saccharomyces s.a.  68.333 × 104 67.358 × 104 69.307 × 104 5.835 5.828 5.841 S pcerevisiae s.a. + ABC 3.000 × 102 2.452 × 102 3.548 × 102 2.477 2.390 2.550 0.12 <0.00051st test  s.a. + B 2.500 × 102 2.000 ×102 3.000 × 102 2.398 2.301 2.477 0.17 <0.0005Saccharomyces s.a. 19.365 × 105 19.306 × 105 19.425 × 105 6.287 6.286 6.288 S pcerevisiae s.a. + ABC 5.500 × 103 5.265 × 103 5.735 × 103 3.740 3.721 3.758 0.10 <0.00052nd test s.a. + B 25.250 × 103 24.748 × 103 25.752 × 103 4.402 4.394 4.411 0.29 <0.0005were obtained from tcS biosciences Ltd. (bo-tolph claydon, buckingham, UK), and are the standards used for quality control testing in the Laboratory “rocchi dr. Eugenio s.r.l”, baz-zano (bologna, Italy). Solid media used included Sabouraud dex-trose agar (biokar diagnostic) for Aspergillus brasiliensis and Saccharomyces cerevisiae, and tryptone soy agar (Oxoid) for all other target microorganisms. Each solid medium was pre-pared in the following different versions: undi-luted, with the addition of 1 mL Abc extract 100 mL-1 of medium, and with the addition of 1 mL b extract 100 mL-1 of medium. testing veri-fied that the addition of extracts did not signif-icantly modify the pH of the medium; the pH remained in the interval of 7.1 ± 0.02 for tryp-tone soy agar, and of 5.5 ± 0.05 for Sabouraud dextrose agar.the stock suspensions for the inoculum were produced by collecting a loopful (with a 1 µL loop) of target microorganisms from slant cul-tures, and dispersing the microorganisms into 10 mL of sterile ringer 1\4 solution (biokar di-agnostic). From these stock suspensions, dec-imal dilutions were prepared up to the third. then 50 µL of the third and the second deci-mal dilutions were used to inoculate four rep-lica plates, undiluted on the surface of the me-dia with a Don Whitley spiral plater. Incubation conditions were chosen in accordance with the nature of each target microorganisms as fol-lows: Aspergillus brasiliensis, 25°c for 5 days; Escherichia coli, 37°c for 24 hours; Staphylo-coccus aureus, 37°c for 48 hours; Listeria mono-cytogenes, 37°c for 48 hours; Bacillus cereus, 30°c for 48 hours; Saccharomyces cerevisiae, 25°c for 5 days.Ital. J. Food Sci., vol. 25 - 2013 241rESULtS AND cONcLUSIONSAt the end of the incubation, a relative count was performed for each microorganism, enabling comparison of the growth on control media with that on media modified by adding the two ex-tracts, Abc and b. the calculations took into ac-count the results obtained from all dilutions and replicas sown with each target microorganism, and applied the formula as per the ISO 8199:2005 standard at point 8.4.2. the 95% confidence in-terval was also determined for each count, as in-dicated by the aforementioned ISO standard. Pre-liminary sterility evaluations were made on media loaded with the extracts Abc and b, and showed that the extracts had negligible bacteria content of 560 cfu mL-1 for the Abc extract and 640 cfu mL-1 for the b extract. Such values confirm the non-existence of interference from microbial con-tent peculiar to the extracts.table 2 reports the results for each target mi-croorganism; values express the growth as colo-ny forming units (cfu) per mL, derived from sow-ing the stock suspensions on either the undi-luted media or the media modified with the two examined yucca extracts. table 2 also presents the data expressed in log cfu mL-1 and the 95% confidence intervals. Data show that yucca ex-tracts had a significant microbial growth reduc-tion effect only on S. Cerevisiae, statistically con-firmed by p-values (<0.0005) as reported in ta-ble 2; it is very clear that the effect of Abc and b extracts doesn’t exist against other target mi-croorganisms at the applied 1% concentration (no inhibition is readable on logarithmic scale). Data clearly show the effects of both extracts on Saccharomyces cerevisiae compared to the un-altered media; the activity of the two Yucca fil-amentosa L. extracts led to inhibition by more than 3 logarithmic scales. the activity is con-firmed by a 2nd test performed ten days later: the results are reported in table 2.the Student’s t-test was used to analyse the data, confirming that a normal distribution could be assumed. this statistical analysis showed that except in the case of S. cerevisiae, non-signifi-cant differences were detected in the target mi-croorganism counts among the undiluted media and the media modified with extracts Abc and b. the statistically significant effect on S. cerevisiae growth was repeatable between the four replicas, in two quantitative tests performed using a basic medium from two different batches. the present reported, specifically growth in-hibitory activity of Yucca filamentosa L. extracts against yeast opens a wide range of possible ap-plications for using these extracts in the food sec-tor. 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First data on the antimicrobial activity of Yucca filamentosa L. bark exytacts

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First data on the antimicrobial activity of Yucca filamentosa L. bark exytacts

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