Peptide Fractions Obtained from Rice By- Products by Means of an Environment- Friendly Process Show In Vitro Health-Related Bioactivities

Recently, the isolation of new health-related bioactive molecules derived from agro-food industrial by-products by means of environment-friendly extraction processes has become of particular interest. In the present study, a protein by-product from the rice starch industry was hydrolysed with five commercial proteolytic enzymes, avoiding the use of solvents or chemicals. The digestion processes were optimised, and the digestates were separated in fractions with four different molecular weight ranges by using a cross-flow membrane filtration technique. Total hydrolysates and fractions were tested in vitro for a wide range of biological activities. For the first time rice-derived peptides were assayed for anti-tyrosinase, anti-inflammatory, cytotoxicity and irritation capacities. Antioxidant and anti-hypertensive activities were also evaluated. Protamex, Alcalase and Neutrase treatments produced peptide fractions with valuable bioactivities without resulting cytotoxic or irritant. Highest levels of bioactivity were detected in Protamex-derived samples, followed by samples treated with Alcalase. Based on the present results, a future direct exploitation of isolated peptide fractions in the nutraceutical, functional food and cosmetic industrial fields may be foreseen

Biomass recycling from a riboflavin cultivation with B. subtilis: lysis, extract production and testing as substrate in riboflavin cultivation

Die industrielle Herstellung diverser Feinchemikalien wie Vitamine und Enzyme mittels Fermentation hat bereits in vielen Fällen die chemische Herstellung abgelöst. Obwohl die in diesen Prozessen produzierte Biomasse ein sehr hohes Energiepotential hat, wird sie meistens als Abfallprodukt entsorgt und in wenigen Fällen als Futtermitteladditiv oder Dünger verwendet. Betrachtet man die Zusammensetzung der Biomasse, so stellt man fest, dass diese Biomasse genau die elementare Zusammensetzung hat, die im Fermentationsmedium benötigt wird. Dies trifft auch auf die fermentative Riboflavinherstellung mittels B. subtilis zu. Ziel der vorliegenden Arbeit war, die aus der Fermentation anfallende Biomasse zu verwerten, indem daraus ein Extrakt hergestellt wird, der wieder in der Fermentation eingesetzt werden kann. Hierdurch könnten die Prozesskosten erheblich gesenkt werden. Der Zellaufschluss zur Extraktherstellung erfolgt mittels Autolyse, deren optimale Bedingungen in einem pH Bereich von pH 6,5 bis 7,5 und einer Temperatur von 40 °C sind. Unter diesen Bedingungen können nach 24 h noch ca. 43 % der ursprünglichen Biotrockenmasse nachgewiesen werden. Da die endogenen Proteasen von B. subtilis für eine entsprechende Extraktqualität nicht ausreichend sind, müssen weitere Proteasen zugegeben werden. Durch ein Screening konnte für den pH-Wert von 6,5 die Kombination Protease N „Amano“, Promod 194P und Peptidase R und für den pH-Wert von 7,5 die Kombination Alcalase, Umimazyme und Protease P „Amano“ festgestellt werden. Die Zugabe der Proteasen bewirkt eine Beschleunigung der Autolyse, eine Erhöhung des Aufschlussgrades und eine Abnahme der Proteinkonzentration im Überstand. Von der ursprünglichen Biotrockenmasse werden bei pH 7,5 noch ca. 25 % und bei pH 6,5 noch ca. 35 % nachgewiesen. Trotz des erhöhten Lysegrades nimmt die Proteinkonzentration im Überstand bei pH 7,5 auf 16 % und bei pH 6,5 auf ca. 25 % der theoretischen Gesamtproteinkonzentration ab. Da ein vollständiger Abbau der freigesetzten hochmolekularen Proteine nicht erreicht wird, werden diese mittels Ultrafiltration (Ausschlussgrenze 10 kDa) entfernt um den erhaltenen Überstand in der Fermentation einsetzen zu können,. Die Induktion der Autolyse direkt nach Fermentationsende bietet einige Vorteile in Bezug auf das eigentliche Produkt Riboflavin. Während der Fermentation wird von B. subtilis der biosynthetische Vorläufer des Riboflavins, das 6,7-Dimethyl-8-Ribityllumazin, in geringen Mengen ins Medium ausgeschieden. Während der Autolyse wird dieses durch die freigesetzte Riboflavinsynthase zu Riboflavin umgesetzt und somit eine Ausbeutesteigerung von bis zu 6 % in Bezug auf die Riboflavin–Konzentration am Ende der Fermentation erzielt. Ein weiterer Vorteil dieser Vorgehensweise betrifft die Reinheit des Produktes Riboflavin. Durch die Zugabe der Proteasen bei der Autolyse, kann der Protein- und DNA-Gehalt signifikant gesenkt werden. In der Fermentation werden mit den Extrakten, die durch direkte Autolyse hergestellt werden, unabhängig vom pH-Wert der Autolyse, Ausbeuten von durchschnittlich 77 % erreicht. Wird vor der Induktion der Autolyse die Biomasse isoliert und gewaschen, so kann eine Ausbeute von durchschnittlich 93 % im Bezug auf die Standardfermentation mit Hefeextrakt erzielt werden. In allen durchgeführten Fermentationen mit den hergestellten Bacillus-Extrakten konnte ein langsameres Wachstum der Biomasse und ein um 1-3,5 h verzögerter Feed–Start im Vergleich zur Fermentation mit Hefeextrakt festgestellt werden. Nähere Untersuchungen hierzu ergeben, dass einerseits Stress Proteine und andererseits eine erhöhte Natriumkonzentration dafür verantwortlich sind.The industrial production of various fine chemicals like vitamins and enzymes by fermentation becomes increasingly important. Although the produced biomass has a very high energy potential, it is in most cases removed as a waste product. Alternatively it can be used as a feedstuff additive, or as a fertilizer. Regarding on the elements of this waste it consists principally exactly of the same elements of which the cells are build. Therefore, it makes sense to develop a process, which allows processing this waste to an extract that can be used in cultivation processes. This also applies to the fermentative riboflavin production. The objective of the present study was to investigate a way by which the produced biomass can be recycled. We considered to evaluate a way of producing an extract of the biomass and reuse it instead of yeast extract in the cultivation. For processing the extract the cells were disrupted by autolysis. The optimal autolysis condition were in the pH range between 6.5 and 7.5 and a temperature of 40 °C. After 24 h approximately 43 % of the original cell dry mass could be detected under these conditions. Since the endogenous proteases of B. subtilis were not sufficient for a corresponding extract quality, additional proteases were added during autolysis. By screening the combination of Protease N "Amano", Promod 194 P and Peptidase R were found to be best suited for pH 6.5. For pH 7.5 the combination Alcalase, Umimazyme and Protease P "Amano" was best suited. Additionally to the decreased protein concentration the proteases also causes an acceleration of autolysis and an increased lysis degree. At pH 7.5 only 25% respectively 35 % at pH 6.5 of the initial cell dry mass could be detected after 24 h. By the addition of proteases the protein concentration in the supernatant decrease to 16 % at pH 7.5 respectively 25 % at pH 6.5 despite a higher lysis degree. A complete breakdown of the released high molecular proteins was not possible. For fermentation trials with the received extract it was necessary to perform a cross flow filtration (cut-off 10 kDa) to remove the remaining high molecular proteins. The induction of autolysis directly after the fermentation offers some advantages regarding to the main product riboflavin. During fermentation B. subtilis releases small amounts of 6,7-dimethyl-8-ribityllumazine, the biosynthetic precursor of riboflavin, to the supernatant. During autolysis 6,7-dimethyl-8-ribityllumazine was converted to riboflavin by the released riboflavin synthase. By this conversion the riboflavin concentration increased up to 6 % of the initial riboflavin concentration. Another advantage of this procedure concerns the purity of the product riboflavin. By the addition of proteases during autolysis, the protein and DNA amount trapped in the crystals can be lowered significantly. In comparison with the fermentation with yeast extract a riboflavin yield of 77 % could be reached with the received Bacillus-extracts. If the biomass was isolated and washed before autolysis a riboflavin yield of 93 % could be reached. During this trials a slower growing of the biomass and a delayed feed-start (1 - 3.5 h) in comparison to the fermentation with yeast extract could be observed. Further investigations showed, that General Stress Proteins and a increased sodium concentration is responsible for the delayed feed-start

Schematic representation of the proposed process.

<p>Rice protein by-product from starch industry was enzymatically hydrolysed by one of the three selected commercial proteases at the defined conditions. The resulting liquid supernatant was fractionated by a sequence of four cross-flow membrane filtrations at different molecular weight cut-offs, leading to the isolation of different peptide ingredients.</p

Mono-dimensional SDS-PAGE (16% w/v acrylamide) protein separation of digestates obtained with different E/S (U/g) ratios.

<p>(<b>A</b>) Alcalase and (<b>B</b>) Protamex hydrolysates after 2 hours of incubation. Loaded volume: 3 μL of total samples, 25 μL of supernatants. Loading scheme: lane 1) molecular weight markers; 2) ND, not digested total sample at room temperature; 3) ND 60°C, not digested total sample 2h at 60°C; 4) 0.5 U/g, total sample; 5) 1 U/g, total sample; 6) 2 U/g, total sample; 7) ND 60°C, not digested supernatant 2h at 60°C; 8) 0.5 U/g, supernatant; 9) 1 U/g, supernatant; 10) 2 U/g, supernatant.</p

<i>In vitro</i> biological activities of the supernatants collected after scale up of protease digestion in flasks.

<p><i>In vitro</i> biological activities of the supernatants collected after scale up of protease digestion in flasks.</p

Protein quantification and biological activities of peptide fractions.

<p>Protein concentration in g/L (A, B, C), antioxidant activity expressed as g of ascorbic acid (AA) per L (D, E, F), anti-hypertensive activity IC50 expressed as g/L (G, H, I, J), and anti-tyrosinase activity expressed as g of kojic acid (KA) per L (K, L, M) on fractions obtained from Neutrase, Alcalase and Protamex hydrolyses. Data represent the mean ± SD (at least n = 3).</p

Characterisation of initial rice by-product.

<p>Characterisation of initial rice by-product.</p

Protein quantification on total and supernatant samples.

<p>Enzymatic hydrolyses (2 hours) with Neutrase (neu), papain (pap), Alcalase (alc), Protamex (pro) and Flavourzyme (flav), were performed. Numbers under the enzyme abbreviations indicate the E/S ratio in U/g (unit of enzyme per g of protein). Results are expressed as g of protein per litre of digestate (g/L) ± SD (at least n = 4).</p