Bioactivity and microbial content of Lippia multiflora leaves, a herbal tea from Ghana

Arthur, Hanson (2009-12)

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2009.

Thesis

ENGLISH ABSTRACT: The consumption of herbal teas is an increasing phenomenon among tea consumers globally. However, herbal teas that are not pre-treated to reduce their microbial load are a health risk to consumers, in spite of their potential health-promoting properties. The aim of this study was to develop a steam pasteurisation treatment to reduce the microbial load on Lippia multiflora Moldenke (Verbanaceae) tea leaves, a herbal tea from Ghana, identify the bacteria present, and to evaluate the effect of the steam treatment on the bioactive constituent of the leaves. An HPLC method was developed and optimised for the identification and quantification of verbascoside, the major antioxidant compound of L. multiflora herbal infusion. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to confirm the presence of the compound in the infusion. Ascorbic acid was used as a stabilising agent during the quantification process to prevent the degradation of verbascoside. The hot water infusion of L. multiflora was compared to those of Aspalathus linearis (rooibos) and Cyclopia spp. (honeybush) on the basis of their soluble solids and total polyphenol contents, as well as on their antioxidant activities. In addition to verbascoside, another compound with the same parent and fragment ions as verbascoside was present in the infusion. A 100 ml infusion of L. multiflora had significantly (P < 0.05) higher soluble solids and total polyphenol contents, and antioxidant activities than those of rooibos and honeybush. The rooibos infusion showed significantly (P < 0.05) higher soluble solids and total polyphenol contents as well as antioxidant activities than honeybush. On the basis of soluble solids, rooibos showed a significantly (P < 0.05) higher total polyphenol content and a lower ferric-reducing activity than L. multiflora. Both teas, however, did not differ significantly with respect to the DPPH antioxidant activity. The effect of steam pasteurisation on the microbial load of L. multiflora herbal tea leaves was evaluated. Five samples of the tea were steam pasteurised at 99.8°C for 2.5 min and five samples were unpasteurised. Microbial enumeration was conducted in duplicate on potato dextrose agar (PDA), plate count agar (PCA), violet red bile agar (VRBA), yeast peptone dextrose agar (YPDA), and de Man Rogosa Sharpe agar (MRS). Morphologically distinct colonies were isolated, sub-cultured and their Gram reaction recorded. These bacteria were identified to the species level using 16S ribosomal DNA (rDNA) sequence data. Most of the bacteria identified belonged to the genus Bacillus. One species each from the genera Pantoea and Kocuria were also identified, but only the Bacillus species survived the steam treatment. Coliform bacteria detected prior to pasteurisation were not detected after steam treatment. Steam pasteurisation reduced the microbial load from 104 to 102 cfu.g-1. The effects of the steam pasteurisation on the soluble solid, total polyphenol, and the active compound contents of L. multiflora, as well as the antioxidant activities were studied. Pasteurisation did not significantly (P > 0.05) change the soluble solids, total polyphenol and active compound contents, or the antioxidant activity. Steam pasteurisation is potentially an effective method to treat L. multiflora herbal teas prior to consumption. However, the steam treatment should complement good agricultural and hygienic practices rather than replace them as some bacteria can survive this treatment. The identification and quantification of verbascoside in L. multiflora infusion, as well as the relatively higher antioxidant contents compared to rooibos and honeybush should provide the basis for future studies on the therapeutic application of this herbal tea. Also, verbascoside could potentially form the basis for future quality control of L. multiflora.

AFRIKAANSE OPSOMMING: Daar is 'n wêreldwye toename in die verbruik van kruietee. Kruietee wat egter nie vooraf-behandelings ontvang om die mikrobiese lading te verlaag nie kan, ten spyte van moontlike gesondheidsvoordele, ook 'n potensiële gesondheidsrisiko vir verbruikers inhou. Die doel van hierdie studie was om 'n stoompasteurisasie-behandeling te ontwikkel wat die mikrobiese lading op Lippia multiflora teeblare, 'n kruietee van Ghana, te verlaag. Verder is die teenwoordige bakterieë geïdentifiseer en die effek van 'n stoombehandeling op die bio-aktiewe komponente in die teeblare is ook geëvalueer. 'n Hoë-druk vloeistof-chromatografie metode is ontwikkel en ge-optimiseer vir die identifikasie en kwantifisering van verbaskosied, 'n hoof antioksidant komponent in L. multiflora kruie aftreksels. Vloeistof chromatografie, gekoppel aan in-lyn massa spektroskopie is ook gebruik om die teenwoordigheid van die komponent in die aftreksel te bevestig. Tydens die kwantifiseringsproses is askorbiensuur as 'n stabiliseringsagent gebruik om die degradasie van verbaskosied te voorkom. Die warm water aftreksel van L. multiflora is vergelyk met die van Aspalathus linearis (rooibos) en Cyclopia spp. (heuningbos) in terme van hul opgeloste vastestof- en totale polifenol inhoude, asook hul antioksidant aktiwiteite. 'n Ander komponent buiten verbaskosied, maar met dieselfde ouer en fragment ione, was ook in die aftreksel teenwoordig. 'n 100 ml L. multiflora aftreksel het beduidend (P < 0.05) meer opgeloste vastestowwe, totale polifenole en antioksidant aktiwiteit getoon as rooibos en heuningbos. Rooibos het weer beduidend (P < 0.05) meer opgeloste vastestowwe, totale polifenole, en antioksidant aktiwiteit as heuningbos. In terme van opgeloste vastestowwe het rooibos 'n beduidende (P < 0.05) hoër totale polifenol inhoud en laer ferriet-reduserende aktiwiteit as L. multiflora. Beide tee het egter nie beduidend verskil ten opsigte van hul antioksidant aktiwiteit nie. Die effek van stoompasteurisasie op die mikrobiese lading van L. multiflora kruieteeblare is geëvalueer. Vyf teemonsters is gestoompasteuriseer by 99.8°C vir 2.5 min en 5 verdere monsters is nie gepasteuriseer nie. Mikrobe-tellings is in Mikrobe-tellings is in v duplikaat op potato dextrose agar (PDA), plate count agar (PCA), violet red bile agar (VRBA), yeast peptone dextrose agar (YPDA), en de Man Rogosa Sharpe agar (MRS) gedoen. Morfologies onderskeibare kolonies is geïsoleer, her-gekweek en hul Gram status genotuleer. Hierdie bakterieë is daarna tot op spesie-vlak geïdentifiseer deur 16S ribosomale DNS (rDNS) volgorde bepalings. Die meerderheid van die geïdentifiseerde bakterieë behoort tot die genus Bacillus en een spesie elk van die genera Pantoea en Kocuria is ook geïdentifiseer. Slegs Bacillus spesies het egter die stoompasteurisasie behandeling oorleef. Kolivorme bakterieë wat voor pasteurisasie waargeneem is was afwesig na die stoom behandeling. Stoompasteurisasie het ook die mikrobiese lading van 104 na 102 kve.g-1 verminder. Die effek van stoompasteurisasie op die opgeloste vastestowwe, totale polifenole en die aktiewe-komponent inhoud van L. multiflora, asook die antioksidant aktiwiteit is bestudeer. Pasteurisasie het die opgeloste vastestowwe, totale polifenole, aktiewe komponente en die antioksidant aktiwiteit nie-beduidend (P > 0.05) verander. Stoompasteurisasie kan potensieël 'n effektiewe metode wees vir die behandeling van L. multiflora kruietee voor verbruik. Die stoombehandeling moet egter saam met goeie landbou- en higiëniese praktyke gebruik word eerder as om dit te vervang aangesien sommige bakterieë hierdie stoombehandeling kan oorleef. Die identifikasie en kwantifisering van verbaskosied in L. multiflora aftreksels, asook die hoër antioksidant inhoud vergeleke met rooibos en heuningbos verskaf moontlikhede vir verder navorsing in die terapeutiese aanwending van hierdie kruietee. Verbaskosied kan ook moontlik die basis vorm vir toekomstige kwaliteitskontrole van L. multiflora.

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