The design and development of healthy products based on probiotics and plant extracts represents an innovative solution capable of combining the beneficial properties for health of these two ingredients. However, the combination of botanical extracts and living microorganisms is a major challenge for formulators due to the known antimicrobial properties that characterize most plant extracts. The possibility of having simple, fast and inexpensive methodologies capable of outlining what the interaction between probiotics and botanicals, which one intends to include in the formulations, offers considerable advantages for the manufacturer, who would obtain preliminary data useful for the design of the product. One of the main objectives of this study is the evaluation of the compatibility between probiotics and botanical extracts, using an in vitro test that exploits the colorimetry of the WST-8 salt, capable of quantifying cellular metabolic activity. The project was conducted by preparing model samples that simulated the direct contact between the probiotic and the botanical extracts, on which the colorimetric test was conducted which made it possible to quantify the metabolic activity of the probiotic cells by UV-Vis spectrophotometry in order to finally derive the percentages of inhibition or promotion due to the presence of the extracts. In addition, prototypes of the finished product, composed of probiotics and plant extracts, were prepared and/or monitored, on which long-term stability studies were conducted in the climatic zone, at T 25°C±2°C and humidity 60%±5%, to seek a correspondence with the colorimetric test. The results of the in vitro test obtained show that the interaction of most of the extracts (propolis, Eleutherococcus senticosus, Astragalus membranaceus, Rhodiola rosea and Trigonella foenum-graecum) with Lactobacillus rhamnosus HN001 HOWARU® (considered as a model probiotic for Lactoacillaceae) is characterized by a biphasic curve, at low concentrations of extract, a portion indicative of promotion of the cellular metabolism of the probiotic followed by a second part, at higher concentrations, reporting inhibition data. Allium sativum extract has been shown to promote the metabolic activity of the tested strain without ever inhibiting it, while extracts such as Hibiscus sabdariffa, D-mannose, Cimicifuga racemosa and Melissa officinalis have caused only a general inhibition. The subsequent comparison of the data obtained with those of the long-term stability studies of some products containing the same extracts and probiotics of the Lactobacillaceae family (Lactobacillus reuteri LRC®, Lactobacillus acidophilus LA-14, Lacticaseibacillus rhamnosus CA-15, Lactobacillus plantarum DR7 and Lactobacillus paracasei LPC37®) made it possible to assess the predictivity of the colorimetric assay, which was around 80%. The in vitro test presents critical issues when complex mixtures of extracts are tested and against Bifidobacteriaceae. In conclusion, the metabolic activity inhibition assay with WST-8 salt optimized for probiotics is concretely able to quantify the influence that certain extracts can have on the metabolism of Lactobacillus rhamnosus HN001. In addition, it has a good success rate in predicting the interactions that occur between probiotics of the Lactobacillaceae family and different botanicals. Further developments of the thesis project will be oriented to extend the applicability to different probiotics and mixtures of extracts, as well as to the application of other analytical techniques, such as flow cytometry.
Il design e lo sviluppo di prodotti salutistici a base di probiotici ed estratti vegetali rappresenta una soluzione innovativa in grado di combinare le proprietà benefiche per la salute di questi due ingredienti. Tuttavia, la combinazione di estratti botanici e microrganismi viventi risulta essere una grande sfida per i formulatori a causa delle note proprietà antimicrobiche che caratterizzano gran parte degli estratti vegetali. La possibilità di avere a disposizione delle metodologie semplici, rapide e poco dispendiose in grado di delineare quale possa essere l’interazione tra i probiotici e i botanicals, che si è intenzionati a inserire nelle formulazioni, offre notevoli vantaggi per il produttore, che otterrebbe dati preliminari utili per il design del prodotto. Uno dei principali obiettivi di questo studio è la valutazione della compatibilità tra probiotici ed estratti botanici, mediante l’utilizzo di un test in vitro che sfrutta la colorimetria del sale WST-8, in grado di quantificare l’attività metabolica cellulare. Il progetto è stato condotto preparando dei campioni modello che simulassero il contatto diretto tra il probiotico e gli estratti botanici, sui quali è stato condotto il test colorimetrico che ha permesso di quantificare mediante spettrofotometria UV-Vis l’attività metabolica delle cellule di probiotico per poter infine ricavare le percentuali di inibizione o promozione dovute alla presenza degli estratti. Inoltre, sono stati preparati e/o monitorati dei prototipi di prodotto finito, composti da probiotici ed estratti, sui quali sono stati condotti studi di stabilità long-term in zona climatica, a T 25°C±2°C e umidità 60%±5%, per cercare una corrispondenza con il test colorimetrico. I risultati del test in vitro ottenuti mostrano che l’interazione della maggior parte degli estratti (propoli, Eleutherococcus senticosus, Astragalus membranaceus, Rhodiola rosea e Trigonella foenum-graecum) con Lactobacillus rhamnosus HN001 HOWARU® (considerato come probiotico modello per le Lactoacillaceae) è caratterizzata da una curva bifasica avente, a basse concentrazioni di estratto, una porzione indicativa di promozione del metabolismo cellulare del probiotico seguita da una seconda parte, a concentrazioni più elevate, che riporta dati di inibizione. L’estratto di Allium sativum, in particolare, ha dimostrato di promuovere l’attività metabolica del ceppo testato senza mai di inibirla, mentre estratti come Hibiscus sabdariffa, D-mannosio, Cimicifuga racemosa e Melissa officinalis ne hanno causato solamente una generale inibizione. La successiva comparazione dei dati ottenuti con quelli degli studi di stabilità a lungo termine di alcuni prodotti contenenti gli stessi estratti e probiotici della famiglia delle Lactobacillaceae (Lactobacillus reuteri LRC®, Lactobacillus acidophilus LA-14, Lacticaseibacillus rhamnosus CA-15, Lactobacillus plantarum DR7 e Lactobacillus paracasei LPC37®) ha permesso di fare una valutazione sulla predittività del saggio colorimetrico, che è risultata attestarsi attorno al 80%. Il test in vitro presenta delle criticità nel momento in cui vengono testate delle miscele complesse di estratti e nei confronti delle Bifidobacteriaceae. In conclusione, il saggio di inibizione dell’attività metabolica con sale WST-8 ottimizzato per i probiotici è concretamente in grado di quantificare l’influenza che determinati estratti possono avere sul metabolismo di Lactobacillus rhamnosus HN001. Inoltre, possiede una buona percentuale di successo nel predire le interazioni che si instaurano tra probiotici della famiglia delle Lactobacillaceae e diversi botanicals. Ulteriori sviluppi del progetto di tesi saranno orientati a estendere l’applicabilità a differenti probiotici e miscele di estratti, nonché all’applicazione di altre tecniche analitiche, come ad esempio la citofluorimetria.
Valutazione dell'interazione tra probiotici ed estratti botanici per il design di integratori alimentari: test in vitro di inibizione dell'attività metabolica
GIACCA, VERONICA
2023/2024
Abstract
The design and development of healthy products based on probiotics and plant extracts represents an innovative solution capable of combining the beneficial properties for health of these two ingredients. However, the combination of botanical extracts and living microorganisms is a major challenge for formulators due to the known antimicrobial properties that characterize most plant extracts. The possibility of having simple, fast and inexpensive methodologies capable of outlining what the interaction between probiotics and botanicals, which one intends to include in the formulations, offers considerable advantages for the manufacturer, who would obtain preliminary data useful for the design of the product. One of the main objectives of this study is the evaluation of the compatibility between probiotics and botanical extracts, using an in vitro test that exploits the colorimetry of the WST-8 salt, capable of quantifying cellular metabolic activity. The project was conducted by preparing model samples that simulated the direct contact between the probiotic and the botanical extracts, on which the colorimetric test was conducted which made it possible to quantify the metabolic activity of the probiotic cells by UV-Vis spectrophotometry in order to finally derive the percentages of inhibition or promotion due to the presence of the extracts. In addition, prototypes of the finished product, composed of probiotics and plant extracts, were prepared and/or monitored, on which long-term stability studies were conducted in the climatic zone, at T 25°C±2°C and humidity 60%±5%, to seek a correspondence with the colorimetric test. The results of the in vitro test obtained show that the interaction of most of the extracts (propolis, Eleutherococcus senticosus, Astragalus membranaceus, Rhodiola rosea and Trigonella foenum-graecum) with Lactobacillus rhamnosus HN001 HOWARU® (considered as a model probiotic for Lactoacillaceae) is characterized by a biphasic curve, at low concentrations of extract, a portion indicative of promotion of the cellular metabolism of the probiotic followed by a second part, at higher concentrations, reporting inhibition data. Allium sativum extract has been shown to promote the metabolic activity of the tested strain without ever inhibiting it, while extracts such as Hibiscus sabdariffa, D-mannose, Cimicifuga racemosa and Melissa officinalis have caused only a general inhibition. The subsequent comparison of the data obtained with those of the long-term stability studies of some products containing the same extracts and probiotics of the Lactobacillaceae family (Lactobacillus reuteri LRC®, Lactobacillus acidophilus LA-14, Lacticaseibacillus rhamnosus CA-15, Lactobacillus plantarum DR7 and Lactobacillus paracasei LPC37®) made it possible to assess the predictivity of the colorimetric assay, which was around 80%. The in vitro test presents critical issues when complex mixtures of extracts are tested and against Bifidobacteriaceae. In conclusion, the metabolic activity inhibition assay with WST-8 salt optimized for probiotics is concretely able to quantify the influence that certain extracts can have on the metabolism of Lactobacillus rhamnosus HN001. In addition, it has a good success rate in predicting the interactions that occur between probiotics of the Lactobacillaceae family and different botanicals. Further developments of the thesis project will be oriented to extend the applicability to different probiotics and mixtures of extracts, as well as to the application of other analytical techniques, such as flow cytometry.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/80626