Use of cysteine endoprotease for reducing cloudiness in drinks
11319516 · 2022-05-03
Assignee
- MALTERIES SOUFFLET (Nogent sur Seine, FR)
- INSTITUT NATIONAL DE RECHERCHE POUR L'AGRICULTURE. L'ALIMENTATION ET L'ENVIRONNEMENT (Paris, FR)
Inventors
- Michel Lopez (Sains en Amienois, FR)
- Myriam Fliss (La Motte Tilly, FR)
- Didier Gilbert Marion (Nantes, FR)
Cpc classification
C12C1/18
CHEMISTRY; METALLURGY
International classification
C12C5/00
CHEMISTRY; METALLURGY
C12C1/18
CHEMISTRY; METALLURGY
A23L2/84
HUMAN NECESSITIES
Abstract
The present invention relates to the use of a cysteine endoprotease or a malt extract to prevent or reduce the cloudiness of a cereal-based beverage, fermented or not.
Claims
1. A method for preparing a fermented cereal-based beverage with increased fermentation yield, comprising treating said fermented cereal-based beverage during its fermentation by adding cysteine endoprotease or a malt extract comprising cysteine endoprotease.
2. The method according to claim 1, wherein the adding of cysteine endoprotease or of a malt extract comprising cysteine endoprotease is at the beginning of the fermentation of the fermented cereal-based beverage being prepared.
3. The method according to claim 1, wherein the malt extract is a processed malt extract that is obtained by recovery and centrifugation filtration of a crude malt extract.
4. The method according to claim 3, wherein the processed malt extract is treated by differential precipitation with ammonium sulphate.
5. The method according to claim 1, wherein the malt extract is a concentrated malt extract.
6. The method according to claim 1, wherein the fermented cereal-based beverage is beer.
7. The method according to claim 6, wherein the beer is selected from the group consisting of low-fermentation beers, high fermentation beers, beers of single, double or triple fermentation, beers with spontaneous fermentation, beers with mixed fermentation, beers prepared with additives, and beers with a range of alcohol levels from 0 to 10%.
8. The method according to claim 1, wherein cysteine endoprotease is selected from the group consisting of cysteine endoprotease A and cysteine endoprotease B of malt.
9. The method according to claim 1 wherein the malt extract is obtained from green malt or dry malt.
10. The method according to claim 1 wherein the malt extract is made from wheat or barley malt.
11. The method according to claim 1, wherein cysteine endoprotease or the malt extract comprising cysteine endoprotease is added in a sufficient amount so that said fermented cereal-based beverage reaches up to 1°P on the Plato gravity scale 8 days after the start of the fermentation.
12. The method according to claim 11, wherein for the equivalent fermentation time, there are fewer fermentable sugars still present in the fermented cereal-based beverage as compared to an identical amount of an identical fermented cereal-based beverage that has not undergone said treatment.
13. A method of reducing the cloudiness of a fermented cereal-based beverage comprising treating said fermented cereal-based beverage by adding during preparation of said fermented cereal-based beverage a cysteine endoprotease or a malt extract comprising cysteine endoprotease; wherein the cysteine endoprotease or the malt extract comprising cysteine endoprotease is added at any stage of the preparation of the fermented cereal-based beverage and is not followed by destroying the cysteine endoproteasic activity.
14. The method according to claim 13, wherein addition of cysteine endoprotease or a malt extract comprising cysteine endoprotease reduces the cloudiness of the fermented cereal-based beverage by at least 65 EBC units when turbidity is measured at −8° C. as compared to an identical fermented cereal-based beverage that has not undergone said treatment.
15. The method according to claim 13, wherein turbidity is measured using the Chapon test.
16. The method according to claim 13, wherein the malt extract is a processed malt extract that is obtained by recovery and centrifugation filtration of a crude malt extract.
17. The method according to claim 16, wherein the processed malt extract is treated by differential precipitation with ammonium sulphate.
18. The method according to claim 13, wherein the malt extract is a concentrated malt extract.
19. The method according to claim 13, wherein the fermented cereal-based beverage is a beer.
20. The method according to claim 19, wherein the beer is selected from the group consisting of low-fermentation beers, high fermentation beers, beers of single, double or triple fermentation, beers with spontaneous fermentation, beers with mixed fermentation, beers prepared with additives, and beers with a range of alcohol levels from 0 to 10%.
21. The method according to claim 13, wherein cysteine endoprotease is selected from the group consisting of cysteine endoprotease A and cysteine endoprotease B of malt.
22. The method according to claim 13, wherein the malt extract is obtained from green malt or dry malt.
23. The method according to claim 13, wherein the malt extract is made from wheat or barley malt.
24. The method according to claim 13, wherein the cysteine endoprotease or a malt extract comprising cysteine endoprotease is added during the fermentation of the fermented cereal-based beverage.
25. The method according to claim 24, wherein the cysteine endoprotease or a malt extract comprising cysteine endoprotease is added during low fermentation, during high fermentation, or during spontaneous fermentation.
26. The method according to claim 24, wherein the adding of cysteine endoprotease or of a malt extract comprising cysteine endoprotease is at the beginning of the fermentation of the fermented cereal-based beverage being prepared.
27. A method of reducing the cloudiness of a fermented cereal-based beverage and increasing fermentation yield of a fermented cereal-based beverage comprising treating said fermented cereal-based beverage during its fermentation by adding cysteine endoprotease or a malt extract comprising cysteine endoprotease.
28. The method according to claim 27, wherein the adding of a cysteine endoprotease or of a malt extract comprising cysteine endoprotease is at the beginning of the fermentation of the fermented cereal-based beverage.
29. The method according to claim 27, wherein cysteine endoprotease or the malt extract comprising cysteine endoprotease is added in a sufficient amount so that said fermented cereal-based beverage reaches up to 1°P on the Plato gravity scale 8 days after the start of the fermentation; and wherein addition of cysteine endoprotease or a malt extract comprising cysteine endoprotease reduces the cloudiness of the fermented cereal-based beverage by at least 65 EBC units when turbidity is measured at −8° C. as compared to an identical fermented cereal-based beverage that has not undergone said treatment.
30. The method according to claim 29, wherein turbidity is measured using the Chapon test.
31. The method according to claim 27, wherein the malt extract is a processed malt extract that is obtained by recovery and centrifugation filtration of a crude malt extract.
32. The method according to claim 31, wherein the processed malt extract is treated by differential precipitation with ammonium sulphate.
33. The method according to claim 27, wherein the malt extract is a concentrated malt extract.
34. The method according to claim 27, wherein the fermented cereal-based beverage is beer.
35. The method according to claim 34, wherein the beer is selected from the group consisting of low-fermentation beers, high fermentation beers, beers of single, double or triple fermentation, beers with spontaneous fermentation, beers with mixed fermentation, beers prepared with additives, and beers with a range of alcohol levels from 0 to 10%.
36. The method according to claim 27, wherein cysteine endoprotease is selected from the group consisting of cysteine endoprotease A and cysteine endoprotease B of malt.
37. The method according to claim 27 wherein the malt extract is obtained from green malt or dry malt.
38. The method according to claim 27 wherein the malt extract is made from wheat or barley malt.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
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DESCRIPTION OF SEQUENCES
(13) TABLE-US-00003 SEQ ID NO: Description 1 Protein sequence of a first variant of barley EP 2 Protein sequence of a second variant of barley EP 3 Protein sequence of a first variant of barley EP 4 Protein sequence of a second variant of barley EP 5 Mature chain of a first variant of barley EP 6 Mature chain of a second variant of barley EP 7 Mature chain of a first variant of the barley EP 8 Mature chain of a second variant of the barley EP
EXAMPLES
Example 1: Decreased Beer Cloudiness with Different Types of Barley Malt
(14) This example shows that an extract of malt obtained from different types of barley may be used to clarify the beer.
(15) Material and Methods
(16) Extraction
(17) 50 g of finished malt of various types of barley were extracted into 200 mL of 0.1 M citrate buffer, pH 4.3, stored in a refrigerator at 4° C. They were blended in a blender for 40 seconds at maximum speed and then centrifuged at 3660 rpm for 10 min at 14° C. The supernatant was recovered and filtered with a filter paper to remove the suspended particles.
(18) Non-Stabilized Beer
(19) The beer used in this example is Fink'bräu Beer 33 cL in a can kept at room temperature (21° C.), filtered on filter paper, and degassed for 20 minutes in an ultrasonic bath.
(20) Samples
(21) The positive control (T+) corresponds to 0.75 mL of Brewers Clarex® diluted 1/25+non-stabilized beer qsp 50 mL.
(22) The negative control (T−) corresponds to 4 mL of 0.1 M citrate buffer pH 4.3+qsp 50 mL of non-stabilized beer.
(23) The samples tested included the following extracts of the barley malts: Metaxa 3086 Ngt 1, Metaxa 3352 Ngt 1, Esterel 23036 B2P1, Esterel 23145 B1PO, Arturio 23005 B2PR, Arturio 23182 B1PO, Azurel 23054 B2PR, Azurel 23108 B2PR, Cervoise 23094 Ngt 1, Cervoise 3453 Ngt 1, Cartel 2965 Ngt 1, Cartel 3259 Ngt 1, Béatrix 23050 B2SA, Béatrix 23189 B2SA, Sébastian 993 Ngt 2, Sébastian 23119 B2P1, Grace 23076 B3ST, Grace 23219 B3ST, Prestige 23077 B3ST, Chill 23151 B3ST, Tipple 23192 B2PR, Tipple 23249 B2SA, Chamonix 404 Ngt 2, Chamonix 3227 Ngt 1, Charmey 436 Ngt 2, Charmey 897 Ngt 2, Henley 3319 Ngt 1, Henley 3328 Ngt 1.
(24) The samples tested consist of 4 mL extract+non-stabilized beer qsp 50 mL.
(25) The mixtures were incubated for 17 h in a water bath at 37° C.
(26) Chapon Test
(27) Controls and samples were incubated at 37° C. for a minimum of 5 h. In transparent plastic test tubes (polystyrene), 0.6 mL of 96% ethanol (=6%) was mixed with qsp 10 mL of sample (×2).
(28) The mixture was incubated for 30 min at −8° C. in a cryostat bath.
(29) Turbidity was measured at −8° C.
(30) Results
(31) The results are shown in
Example 2: Reduction of Beer Cloudiness with Wheat Malt
(32) This example shows that a wheat malt extract may be used to clarify the beer.
(33) Material and Methods
(34) Extraction
(35) 50 g of finished malt of various types of barley were extracted into 200 mL of 0.1 M citrate buffer, pH 4.3, stored in a refrigerator at 4° C. They were blended in a blender for 40 seconds at maximum speed and then centrifuged at 3660 rpm for 10 min at 14° C. The supernatant was recovered and filtered with a filter paper to remove the suspended particles.
(36) Non-Stabilized Beer
(37) The beer used in this example is Fink'bräu Beer 33 cL in a can kept at room temperature (21° C.), filtered on filter paper, and degassed for 20 minutes in an ultrasonic bath.
(38) Samples
(39) The positive control (T+) corresponds to 0.75 mL of Brewers Clarex® diluted 1/25+non-stabilized beer qsp 50 mL.
(40) The negative control (T−) corresponds to 4 mL of 0.1 M citrate buffer pH 4.3+qsp 50 mL of non-stabilized beer.
(41) The samples tested include the following barley malt extracts: barley malt Chill 22304 B2BR, barley malt Arturio 23175 B1CA, barley malt diastasic Arturio 23112 B2BR, wheat malt Apache 23086 (Arcis sur Aube) and wheat malt Bagou (Arcis sur Aube).
(42) The samples tested consist of 0.5, 1, 2, 3 or 4 mL extract+non-stabilized beer qsp 50 mL.
(43) The mixtures were incubated for 17 h in a water bath at 37° C.
(44) Chapon Test
(45) The controls and samples were incubated at 37° C. for a minimum of 5 h. In transparent plastic test tubes (polystyrene), 0.6 mL of 96% ethanol (=6%) was mixed with qsp 10 mL of sample (×2).
(46) The mixture was incubated for 30 min at −8° C. in a cryostat bath.
(47) Turbidity was measured at −8° C.
(48) Results
(49) The results are shown in
Example 3: Identification in the Malt Extract of Enzymes Responsible for the Effect on Turbidity
(50) This example shows the effect of malt extracts on beer cloudiness and the involvement of cysteine barley endoproteases in this effect.
(51) Materials and Methods
(52) Materials
(53) The malt used for the various experiments was obtained from barley grains of the variety Beatrix (Hordeum vulgare) malted by the Soufflet malt (Nogent sur Seine/Aube) but the enzymes are present in all varieties of barley whether they are of winter (two or six rows) or spring.
(54) The malt was ground in a vibrating feeder L 24 from the company Fritsch (Germany).
(55) The various products used for electrophoresis buffers and gels come from the suppliers Alfa Aesar, Carbo Erba and Sigma. The 40% acrylamide comes from Fisher Scientific.
(56) The colorimetric determination of the proteins was carried out with the BC Assay kit from Uptima Interchim.
(57) The ion exchange chromatographs were performed on FPLC Akta explorer 100 and Akta Prime (GE Healthcare, USA) with XK 26/10 columns and Sepharose SP Fast Flow (FF) 50 ml gels and Sepharose DEAE FF (GE Healthcare, USA). Separation is also feasible on other types of cation and anion exchange columns.
(58) The filtration gels were performed on FPLC Akta explorer 10 (Amersham Pharmacia Biotech) and Akta explorer 100 (GE Healthcare, USA) with Sephadex HR (High Resolution) S200 (16/60) and Superose 12 (10/30) columns.
(59) Protein detection was performed by 3-wavelength spectroscopy: at 215, 260 and 280 nm.
(60) HPLC analyzes were carried out on an Alliance Waters 2795 separation module/Waters 2487 dual λ absorbance detector and the column used was a Luna 5μ (C18) 100 Å (250×4.60 mm) (Phenomenex). Detection of the proteins was carried out by 2-wavelength spectroscopy: at 214 and 280 nm.
(61) The samples were analyzed by mass spectrometry, after tryptic hydrolysis, on nano LC-MS/MS ESI ORBITRAP Velos. The peptide analyses were carried out using the Mascot 2.2 software.
(62) The modeling of the enzymes was carried out using the Modeller 9.14 software.
(63) The centrifuge is a Beckman Avanti (USA) J26 XP (rotor: FiberLite® F10BCI-6x500y).
(64) The cloudiness was measured on a Pfeuffer tannometer (Germany) after passing through a Hubert Variostat CC immersion cryothermostat. The non-stabilized control beer is Fink'bräu (Lidl) and the positive control is Brewers Clarex® (DSM, The Netherlands).
(65) The gliadins (total) and the β-lactoglobulins used for the hydrolysis tests were supplied and purified by the INRA of Nantes (BIA center Angers-Nantes/Loire Atlantique).
(66) The peptide Z-GP-pNA and the peptide Z-FR-pNA originate from the company Bachem (Switzerland).
(67) The OD was read by an Epoch plate reader (Biotek, USA).
(68) The dialysis membranes were visking membranes (Medicell Int.) with a cutoff threshold of 12-14000 Da.
(69) Preparation of Buffers
(70) The 0.1M citrate buffer pH 4.3 was prepared with 0.1M citric acid (MW=210.14 g/mol L.sup.−1) and adjusted to pH 4.3 by the addition of citrate sodium to 0.1 M (MW=294.1 g/mol L.sup.−1). For the other citrate buffers used, they were also prepared with the solutions at the indicated molarity and adjusted to the desired pH by addition of sodium citrate.
(71) The 50 mM acetate buffer pH 4 was prepared with 50 mM acetic acid and adjusted to pH 4 by the addition of 50 mM sodium acetate (MW=136.08 g/mol L.sup.−1).
(72) The 50 mM Tris/HCl pH 7.5 buffer was prepared with 50 mM Tris and adjusted to pH 7.5 by the addition of hydrochloric acid (HCl).
(73) The citric acid buffer 0.1 M/disodium phosphate 0.2 M (MW=177.99 g/mol L.sup.−1) was prepared with 0.1M citric acid and adjusted to pH 4 (or 5) by the addition of 0.2M disodium phosphate.
(74) The buffer for the lyophilization test was prepared with 100 mM Tris/HCl pH 8.5 (PM T5ris=121.14 g/mol L.sup.−1) to which was added EDTA (Ethylene diamine tetra acetic 5 mM (MW=292.24 g/mol L.sup.−1), 2 mM cysteine (MW=121.2 g/mol L.sup.−1), 4% mannitol (w/v) and 1% sucrose (w/v).
(75) Preparation of the Extract
(76) The malt grains were ground under liquid nitrogen to 0.5 mm. 48 g of MSi (Initial Dry Material) flour were suspended in 200 mL of 0.1 M citrate buffer pH 4.3 with magnetic stirring at 7° C. for 30 minutes. After centrifugation at 10,000 rpm, for 20 minutes at 7° C., the supernatant was recovered and filtered on filter paper. It is referred to as “crude extract” (EB). This EB was treated with 26% saturation with ammonium sulphate ((NH.sub.4).sub.2SO.sub.4) (SA) or 15% w/v with magnetic stirring at 7° C. for 30 minutes. This followed a second centrifugation at 10,000 rpm for 20 minutes at 7° C. The supernatant was recovered and filtered on filter paper. Thus, the “supernatant treated with 15% ammonium sulfate” (SN-15%-SA) was obtained. SA was added to the SN-15%-SA so as to reach 89% saturation or 65% w/v final. After 1 hour 30 minutes at 7° C. with magnetic stirring at 7° C., the extract was centrifuged again at 10 000 rpm for 20 minutes at 7° C. The supernatant was removed and the base obtained was resuspended in 50 mL of 0.1 M citrate buffer pH 4.3 and dialysed first of all for 30 minutes in 2 L of distilled water followed by two baths of 2 h each in 2 L of 50 mM citrate buffer pH 5 and then overnight against 5 L of 50 mM citrate buffer pH 5 in order to obtain the final extract called C65% SA dia pH 5 (base 65% dialysed ammonium sulphate pH 5) which is also the “forward column” (AC) for passage on the SP Sepharose FF (
(77) Chapon Test
(78) All the efficacy tests for reducing the turbidity of the beer were carried out via the Chapon test (Chapon (1993) J. Inst, Brew 99: 49-56). 2 mL of sample were mixed with 25 mL qs of non-stabilized beer and then incubated at 37° C. for a minimum of 5 h in a water bath. In polystyrene test tubes, 9.4 mL of incubated sample was taken and 0.6 mL of 96% ethanol was added. The tests were carried out in duplicate. After incubation for 30 min at −8° C. in a cryostat bath, the turbidity was measured at −8° C. on a Pfeuffer tannometer. The results are expressed in EBC (European Brewery Convention) (0.25 EBC=1 NTU (Nephelos Turbidity Unit)).
(79) Ion Exchange Chromatographies
(80) Cation exchange chromatography—In a first step, the extract (C65% SA dialyzed at pH 5) was placed on a column (XK 26/10) Sepharose SP FF equilibrated in 50 mM citrate buffer pH 5 (=tp A), flow=13 ml/min (147 cm/h). Elution was carried out by increasing the ionic strength by a linear gradient from 0 to 50% of Buffer B (tp A+1 M NaCl) in 25 CV (column volume). After a plateau at 50% of B for 2 CV, the column was rinsed at 100% B in 7 CV and then rebalanced in buffer A. 15 ml fractions were collected. Only the “non-retained” fraction active in the Chapon test was recovered (NR SP).
(81) Anion Exchange Chromatography
(82) After dialysis twice 2 h and then 1 night in 5 L baths of 50 mM Tris buffer, pH 7.5 (=tp A2), the NR SP was deposited on a DEAE FF Sepharose column, equilibrated by buffer A2, flow rate=5 ml/min and elution was carried out by a linear gradient of 0 to 30% B2 buffer (50 mM Tris pH 7.5+1 M NaCl) over 80 min, followed by a stage of 10 min at 30% and a stage of 20 min at 100% of buffer B2. The collected fractions were 2 mL.
(83) Gel Filtration
(84) Column Sephadex S200—5 mL (4.4 mg of proteins) of the SP/DEAE fraction containing EP-B was deposited on the Sephadex S200 column. The flow rate was 0.5 mL/min and the buffer used was 50 mM citrate buffer pH 4.5. The collected fractions were 5 mL.
(85) Column Superose 12—1.5 mL (1.75 mg protein) of the SP/DEAE fraction containing EP-A was deposited on the Sephadex S200 column. The fraction was previously concentrated on an Amicon Ultra 10 KDa cell of 0.5 mL (Millipore, Ireland). The flow rate was 0.8 mL/min and the buffer used was 50 mM citrate buffer pH 4.5. The collected fractions were 2 mL.
(86) Electrophoresis (SDS-PAGE, PAGE, Zymograms, Tricine)
(87) SDS-PAGE. The different samples were analyzed in SDS-PAGE with a 12% acrylamide separation gel and a 5% concentration gel according to the Laemmli technique (Laemmli (1970) Nature 227: 680-685). The migration was carried out at ambient temperature in an Atto vertical electrophoresis cell under 10 mA in the concentration gel and then 20 mA in the separation gel. Depending on the gels, the colors were made with either Coomassie R250 blue or Coomassie G250 blue. In the wells, 5 μL of known molecular weight standards and 20 μL of sample were deposited. The samples were prepared as follows: 40 μL of sample+20 μL of denaturation solution (50 mM Tris pH 6.8, 20% glycerol, 4% SDS, 0.01% bromophenol blue)+3 μL of β-mercapto-ethanol.
(88) Native PAGE—The protocol was the same as for PAGE gels except for the presence of SDS.
(89) SDS-Tricine PAGE—The SDS-tricine PAGE was performed according to the protocol of Schägger and von Jagow (1987) Anal. Biochem. 166: 368-379, on 10% acrylamide gels for separation before analysis of the bands by mass spectrometry, or 16% for the study of the hydrolysis of the hordeins. The gels were stained with Coomassie G250 blue.
(90) Zymograms—The zymograms were performed on 12% PAGE gels in which 0.1% gliadins was incorporated according to the protocol of Prabucka and Bielawski (2004) Acta Physiol. Plant. 26: 383-391. Before staining with Coomassie R 250 blue, the gel was rinsed twice for 1 hour in 0.1 M citrate buffer pH 4.3 and then incubated in 0.1 M citrate buffer pH 4.3/4 mM cysteine 40° C. overnight.
(91) Analysis in Mass Spectrometry
(92) After SDS-Tricine-PAGE and staining with Coomassie G250 blue, the bands of interest were cut with a cutter under sterile conditions and then analyzed by the BIBS platform of the INRA of Nantes (http://www.bibs.inra.com).
(93) Hydrolysis of Hordeins, Gliadins and β-Lactoglobulins
(94) Hydrolysis of the hordeins: extraction—25 g of flour of barley grains ground to 0.5 mm were mixed with magnetic stirring with 200 ml of 0.5 M NaCl for 1 hour at room temperature. The mixture was then centrifuged at 10,000 rpm for 30 minutes at 12° C. The supernatant was removed and the base was suspended in 200 ml of 0.5 M NaCl and stirred for 1 h at room temperature. After being centrifuged at 10,000 rpm for 30 minutes at 12° C., the supernatant was removed and the base was frozen at −20° C. and then lyophilized.
(95) The lyophilisate was resuspended in 50% propanol (15 mL/g) at 60° C. with magnetic stirring for 45 min and then centrifuged at 10,000 rpm for 30 minutes at 12° C. The supernatant called “supernatant propanol” was stored at 4° C. and the base was extracted again with 50% propanol.
(96) The base was then suspended in 50% propanol (15 mL/g)+2% of β-mercaptoethanol (βME) at 60° C. with magnetic stirring for 45 minutes. It was then centrifuged at 10,000 rpm for 30 minutes at 12° C. The supernatant named “supernatant propanol/β-mercaptoethanol” was stored at 4° C. The base underwent this step twice more.
(97) The propanol and propanol/βME supernatants were dialyzed separately against 20 L of distilled water (3 baths of 2 h and overnight) and then centrifuged at 10,000 rpm for 30 minutes at 12° C.
(98) The bases as well as the supernatants were frozen at −20° C. and then lyophilized. The lyophilisates were then ground to a fine powder and stored at −20° C.
(99) At the end of the extraction, Propanol (H) and Propanol/β-mercaptoethanol Hordeins (HPβME) were obtained (Koehler and Ho (1990) Plant Physiol. Inst Brew 98: 471-478, Zhang and Jones (1996) Planta 199: 565-572).
(100) Since these extracts were difficult to dissolve, they were delipidated before analysis. The HP were delipidized according to the following protocol: in a 15 mL glass tube, 0.302 g of HP were weighed and suspended in 10 mL of dichloromethane+5 mL of acetone. After vortexing for 20 minutes and centrifuging at 4° C. for 10 minutes at 3000 rpm, the supernatant was removed and the base was treated a second time and then dried under vacuum and stored at −20° C. The extract obtained is called HPd1.
(101) Hydrolysis of hordeins: SDS-PAGE and HPLC analysis—After suspending the hordeins (HPdl) (1 mg/mL) in 20 mM sodium-succinate buffer pH 4.5 containing 10 mM β-mercaptoethanol, 2 μg of proteins were added for the fraction containing EP-B as well as for the positive control (Brewer Clarex®). Incubation was carried out at 40° C. 20 μL of the reaction mixture were taken at different times (T=0/5/15/30/60/90 min/2 h/4 h and 24 h), diluted in the denaturation buffer, boiled to stop the reaction and then frozen at −20° C. before analysis on an SDS-Tricine-PAGE 16% gel. In the case of the analysis by reversed phase HPLC, 100 μl of the 24 h extract were taken.
(102) Hydrolysis of Gliadins and β-Lactoglobulin: Reverse-Phase HPLC Analysis. In the case of gliadins and β-lactoglobulin, a 1 mg/mL solution was prepared in 50 mM acetate buffer pH 4. Tests were also carried out by adding DL-Dithiothreitol (DTT) to a final concentration of 2 mM prior to incubation. To initiate the hydrolysis reaction, 2 and 5 μg of proteins (EP-B/EP-A and BC) were added in 200 μL of substrate+/−4 μL of 0.1 M DTT. The reaction mixture incubated at 40° C. for 20 h.
(103) HPLC (High Performance Liquid Chromatography) Reverse Phase
(104) The Luna C18 column (Phenomenex) was equilibrated by eluent A (H.sub.2O milliQ+trifluoroacetic acid (TFA) 0.11%) and elution was carried out by a linear gradient of 0 to 50% eluent B (acetonitrile+0.09% TFA) in 54 min at a flow rate of 1 mL/min for the hydrolysis of gliadins and β-lactoglobulins and a linear gradient from 0 to 60% B in 32 min at a flow rate of 0.8 mL/min for hydrolysis of the hordeins.
(105) 50 μL of sample were injected. The reading was made at 214 nm and 280 nm and the column temperature was maintained at 50° C. (Marchylo and Kruger (1984) Cereal Chem., 61: 295-301, Marchylo et al., (1986) Cereal Chem. 63: 219-231).
(106) Hydrolysis of Z-Gly-Pro-pNA and Z-Phe-Arg-pNA Peptides
(107) For each peptide tested, hydrolysis was carried out at pH 4 and pH 5 (Davy et al., (1998) Plant Physiol., 117: 255-261, Simpson et al., (2001) Plant Sci 161: 825-838).
(108) Hydrolysis of the Z-Gly-Pro-pNA Peptide (Z-GP-pNA)—17.6 μg of proteins were reacted for each enzyme, EP-A, EP-B and BC with qsp 500 μL of acid buffer 0.1 M citric acid/0.2 M disodium phosphate (pH 4 or 5) and 125 μL of 10 mM substrate (solubilized in the same buffer+40% dioxane). The final concentration for the Reaction Volume (VR) in substrate was therefore 2 mM and 8% dioxane.
(109) For each sample, the hydrolysis was done in duplicate (deposition of 250 μL of the VR in a microplate of 96 wells).
(110) The release of the pNA was followed by reading the OD at 410 nm at different times (T=0/10/20/30/40/50/60 min and 1 h30/2 h/2 h30/3 h/3 h30/4 h/4 h30/5 h/5 h30/6 h and 24 h).
(111) Hydrolysis of the Z-Phe-Arg-pNA Peptide (Z-FR-pNA)—17.6 μg of proteins were reacted for each enzyme, EP-A, EP-B and BC, with qsp 500 μL of acetate buffer 50 mM (pH 4 or 5)+5% DMSO and 125 μL of 10 mM substrate solubilized in the same buffer. The final concentration for the reaction volume (VR) in substrate was therefore 2 mM.
(112) For each sample, the hydrolysis was done in duplicate (deposition of 250 μL of the VR in a microplate of 96 wells).
(113) The release of the pNA was monitored by reading the OD at 410 nm at different times (T=0/10/20/30/40/50/60 min and 1 h30/2 h/2 h30/3 h/3 h30/4 h/4 h30/5 h/5 h30/6 h and 24 h).
(114) Freeze-Drying Test
(115) After passing the SP then DEAE columns of the malt extract, the fractions corresponding to the separation peak of the enzyme EP-B were combined. The final volume obtained was divided into two and then separately dialyzed for 30 minutes in distilled water followed by two 2 h baths and then overnight at 4° C. in 50 mM citrate buffer, pH 4.2, for the one and in Tris/HCl/EDTA/Cysteine/Mannitol/Sucrose for the other.
(116) The volume after dialysis was again divided into two. The first portion was stored at −20° C. awaiting analysis in the Chapon test and the second was freeze-dried. The lyophilisate was suspended in a volume of distilled water equal to the initial volume placed on the lyophilizer and was analyzed in the Chapon test.
(117) Results
(118) Comparison in Chapon Test of the Efficacy of the Extracts Obtained at Each Stage of the Enzyme Extraction of Barley Malt
(119) By measuring the decrease of the cloudiness, the Chapon test allows the selection of the active fractions during the purification. Thus, after fractionation with ammonium sulfate, the inventors showed that the activity was conserved in the dialysed SN15% SA and then in the dialysed C65% SA (
(120) Separation of Enzymes from Barley Malt by Ion Exchange Chromatography
(121) The analysis by the Chapon test of the fractions eluted after chromatography of the malt extract (C65% SA dialyzed with pH 5) on a cation exchange column (SP) showed that the enzymatic activity was in the fraction not retained on the column. This fraction was then separated on an anion exchange column (DEAE). This second chromatography made it possible to isolate two enzymatic fractions capable of reducing the turbidity of the beer at 5.2 and 6.1 EBC (
(122) Analysis by Electrophoresis (SDS-PAGE, PAGE, Zymograms)
(123) Analysis by electrophoresis showed that fraction 31 was composed of several protein bands including a main group at about 35 kDa, a band at about 60 kDa, and a thinner band at around 30 kDa. Fraction 46 contained 2 bands of PM at about 60 and 35 kDa. The zymogram confirmed the presence in fractions 31 and 46/47 of enzymes capable of hydrolyzing proteins rich in prolines (gliadins). The enzymes in these two fractions had different mobilities indicating that they were different.
(124) Separation of Enzymes from Barley Malt by Gel Filtration
(125) After separating the enzymes of interest from barley malt by ion-exchange chromatography, the inventors used gel filtration chromatography in order to refine the purity of the fractions exhibiting the enzymatic activity. Separation of fraction 31 on the Sephadex S200 column showed a main peak preceded by two shoulders. Chapon and zymogram analysis revealed that the enzyme was present in the 1C4 fraction corresponding to the main peak. By SDS electrophoresis, the inventors found that this fraction consisted of several protein bands in the region of 35 kDa.
(126) The chromatogram of fraction 47 showed several poorly separated peaks. The Chapon and zymogram analysis revealed that the enzyme was present in the 5A5 fraction corresponding to the first peak.
(127) Characterization of Enzymes by Mass Spectrometry
(128) After SDS-PAGE, the protein bands of fractions 31 and 46 were cut out and analyzed by mass spectrometry after tryptic hydrolysis. In the case of fraction 31, the 2 bands of PM 30 kDa and 28 kDa correspond to the mature form of a cysteine protease: EP-B (Reference Uniprot: P25249 (SEQ ID NO: 3) or P25250: 4)). Two variants of this protease have been described but the differences between these two variants are too small for the analysis to indicate which variant is present. EP-B P25249 and P25250 are proteases of PM 25180 and 25318 and pl 4.77 and 4.96. Fraction 31 also contains β-amylases (PM band 60,000) and serpins (group of bands around 35,000).
(129) In the case of fraction 46, cysteine protease was also found in 2 bands of PM 30 and 28 kDa which correspond to the mature form of cysteine protease EP-A (Uniprot reference: 004675 (SEQ ID NO: 1) or 004677 (SEQ ID NO: 2)). As for EP-B, two variants exist but the analysis does not make it possible to say which variant is present. Based on the mature protein sequences, these proteases had respective PMs of 25590 and 25648 and pls of 4.4 and 4.37. The other bands observed on the electrophoregram also correspond to a β-amylase and serpins.
(130) Sequence alignment showed that the sequences of the two EP-Bs and those of the two EP-As showed 98% and 99% identical amino acids, respectively. In contrast, EP-A had 52% sequential homology with EP-B. These two cysteine proteinases have a similar structure and have an identical catalytic site composed of three amino acids: cysteine (28), aspartate (162) and histidine (163). Although they have a similar effect on decreased beer cloudiness compared to the positive control enzyme (Brewers Clarex®), they are no less different. Indeed, they have only 9 and 11% (respectively) of identical amino acids with Brewers Clarex®. Moreover, the large number of gaps required for this alignment shows that these enzymes have no structural kinship. This lack of structural homology may also be demonstrated with the 3D modeling of the enzyme.
(131) Hydrolysis of Hordeins, Gliadins and β-Lactoglobulins
(132) Electrophoresis analysis of hydrolysis of hordeins—Monitoring of hydrolysis of hordeins by SDS-Tricine-PAGE gel analysis did not allow us to conclude whether there was a real difference in mechanism of action between EP-B and positive control, although EP-B appears to be more effective. Indeed, there are no more bands visible after 24 h of reaction whereas bands may still be observed after 24 h in the presence of Brewers Clarex®.
(133) HPLC analysis of horde hydrolysis—The fraction enriched in EP-B (fraction 31) is capable of hydrolyzing the hordeins and degrades them differently from the positive control (Brewers Clarex®). Indeed, the chromatograms showed that some peptides generated were different.
(134) HPLC analysis of gliadin hydrolysis—In the case of gliadins, chromatograms showed a different action between barley malt enzymes (EP-B and EP-A) and the positive control (Brewers Clarex®). It was found that hydrolysis (in the presence of DTT) by cysteine proteinases is complete, unlike that with Brewers Clarex (BC). Very many hydrophilic peptides have been generated by the hydrolysis by the different enzymes making it difficult to compare the chromatograms. We may still see some differences between the Brewers Clarex and the two proteases of barley malt and between the two proteases (
(135) HPLC analysis of β-lactoglobulin hydrolysis—In the case of hydrolysis of β-lactoglobulins, chromatograms also showed a different action between the enzymes of barley malt (EP-B and EP-A) and the positive control (Brewers Clarex®). This difference is visible with or without DTT (
(136) Hydrolysis of Z-Gly-Pro-pNA and Z-Phe-Arg-pNA Peptides Hydrolysis of Z-Gly-Pro-pNA Peptide—Only Brewers Clarex® was capable of hydrolyzing the Z-GP-pNA peptide in the tests performed (
(137) This experiment demonstrates that the site of action of the Brewers Clarex is different from that of the cysteine proteases EP-A and EP-B.
(138) Hydrolysis of the Z-Phe-Arg-pNA Peptide—In the case of the Z-FR-pNA peptide, the hydrolysis is only visible with the EP-B enzyme. There is no degradation with EP-A or Brewers Clarex. In addition, EP-B is more effective at pH 4. Indeed, the OD at pH 5 is half that of pH 4.
(139) This test leads to the conclusion that barley malt enzymes not only have a different breeding site but also differ from Brewers Clarex (
(140) Freeze-Drying Test
(141) The inventors observed that the enzymatic activity was preserved after lyophilization.
(142) The two buffers tested allow the activity to be maintained, but Tris/EDTA/Cysteine/mannitol/sucrose buffer is more effective.
(143) Conclusions
(144) The inventors have shown that two enzymatic fractions isolated from barley malt are capable of reducing the colloidal cloudiness of beer and of hydrolyzing the prolamins of cereals (hordeins and gliadins). These two enzyme fractions are enriched respectively in two proteases EP-B and EP-A, cysteine proteinases of barley. These two enzymes are very similar in terms of their amino acid sequences (primary structure) and their sequences are not at all identical with that of the Brewers Clarex® enzyme. Finally, the hydrolysis of prolamins (hordeins, gliadins) as well as β-lactoglobulin, as well as the hydrolysis of the Z-Gly-Pro-pNA and Z-Phe-Arg-pNA peptides, show that these cysteine proteinases have different specificities (cleavage sites) and are also different from that of the Brewers Clarex® enzyme.
Example 4: Hydrolysis of Gliadins by EP-A, EP-B and Crude Extract of Malt
(145) This example shows that the enzymes EP-A and EP-B cleave the gliadins in a different way, suggesting a synergy in the action of the two enzymes.
(146) Material and Methods
(147) The gliadins are solubilized at 4 mg/ml in 50 mM acetic acid and then diluted to 1 mg/ml with 50 mM sodium acetate buffer at pH 5 and mixed with various extracts. The mixture of gliadins and acetate buffer represents the control T, the mixture of gliadins and crude extract obtained as described in Example 3 is denoted EB, the mixture of gliadins and EP-A obtained as described in Example 3 is denoted EP-A, and the mixture of gliadins and EP-B obtained as described in Example 3 is denoted EP-B. The mixtures are incubated overnight at 37° C. with stirring.
(148) The amounts of enzymes added were calculated in order to obtain a substrate enzyme ratio of 4%. The samples are incubated for 18 hours at 37° C. before being analyzed by high performance liquid chromatography (HPLC).
(149) Reverse Phase HPLC Analysis:
(150) Reverse phase column: Luna, C18, 100A, 5 μm, 250×4.6 mm (Phenomenex)
(151) Eluent A: H.sub.2O, TFA 0.11%
(152) Eluent B: Acetonitrile, TFA 0.09%
(153) Flow rate 1 ml/min; spectrophotometer detection at 214 and 280 nm. The 280 nm measurement makes it possible to specifically detect proteins containing aromatic amino acids such as tryptophan. At 214 nm, the signal, although less specific, is about 10 times greater than at 280 nm.
(154) The elution was carried out by a linear gradient of 10 to 60% of eluent B in 20 min. Injection volume: 40 μL. Absorbance is recorded at 214 and 280 nm using a Waters 2487 detector.
(155) Results
(156) The hydrolysis tests are carried out on total gliadins. The 2 main peaks corresponding to the gliadins (elution time 16.1 and 17.5 min) show that the gliadins are partially hydrolyzed by the crude extract (EB) and the 2 enzymes. The peaks appearing on the chromatograms after hydrolysis by EP-B and EP-A are different, indicating different cleavage sites (
Example 5: Fermentation Tests in EBC Tubes (“KIRIN” Type)
(157) This example shows the effect of increasing the fermentation yield of the malt extract according to the invention.
(158) Protocols for the Preparation of Barley Malt Extracts
(159) Grinding of Malt
(160) Malt 2RP Pilsen was stored at 4° C. and then crushed on a ZM 200 mill with a 0.5 mm grid.
(161) Extraction
(162) For each extraction, 900 g of malt are used with 3.6 L of citrate buffer. It lasts 30 min at 7° C. with stirring at 250 rpm. The extracts obtained are centrifuged for 25 min at 8400 rpm and at a temperature of 4° C. with an acceleration and deceleration of 3. The supernatant is recovered and roughly filtered on Marcherey Nagel 614 ¼ 320 mm diameter filters.
(163) Deactivation for Denatured Crude Extract (EBD)
(164) The supernatant is heated without stirring until the first broths appear, and then the supernatant is centrifuged for 25 min at 8400 rpm at a temperature of 4° C. and filtered roughly.
(165) Precipitation 15% (w/v) to Ammonium Sulphate (SA)
(166) The precipitation by the addition of ammonium sulphate is carried out under the same conditions as the extraction, with stirring of 250 rpm and at 7° C. The mixture is centrifuged for 25 min at 8400 rpm and at 4° C. The supernatant is recovered and its volume is measured.
(167) Precipitation 65% (w/v) at SA
(168) The base is recovered in 600 ml of citrate buffer and then filtered roughly before filtration on a 0.45 μm filter. The product obtained is stored at 4° C. awaiting desalting.
(169) Desalination
(170) The desalination is carried out on a Sephadex G25 gel packed in an AxiChrom 70 column (GE Healthcare). The device used is the AKTA Prime Plus (GE Healthcare). The desalination is carried out at ambient temperature and the sample is injected with a flow rate of 40 mL/min. The elution is carried out with 0.1M citrate buffer pH 4.3.
(171) Ion Exchange Chromatography
(172) The gel used is the Sepharose Fast Flow gel, equilibrated with 50 mM citrate buffer pH5, the chromatography is carried out at room temperature. The cysteine endoprotease is in the eluted fraction.
(173) Fermentation Tests
(174) An 11°Plato wort is produced by brewing a “EBC congress” type: heating at 45° C. for 30 minutes, followed by a rise in temperature to 70° C. for 60 minutes, then 210 g of malt are poured for 1260 mL of water.
(175) The different additions (malt extract, controls . . . ) are done before seeding in 770 mL of wort, except the PVPP (40 g/HL), which is added before centrifugation.
(176) The modalities tested respectively correspond to: T—is a crude extract of barley malt denatured by heat T.sub.pvpp: no addition TBC is the positive control with addition of Brewers Clarex® EB is the crude extract of barley malt S15 is an extract of malt treated with a solution of 15% ammonium sulphate S65 is a crude extract treated with 15% ammonium sulfate solution and then the supernatant is treated with a solution of 50% ammonium sulphate 65CH is the modality S65, purified on column SP.
(177) The numbers 1, 2 and 3 correspond to 3 volumes of different additions tested: volumes 1 range from 15 to 30 mL, volumes 2 are 22.5 and 30 mL, volume 3 corresponds to 30 or 37.5 mL.
(178) Seeding: for fermentations: 720 mL with dry yeast to measure the Apparent Limit Attenuation: 50 mL with dry yeast.
(179) The fermentation/ripening is carried out at a temperature of 13° C. for about 9 days.
(180) The yeast is purged, then the wort is kept cold (about 2° C., minimum 5 days).
(181) At the end of the storage, the fermented worts are centrifuged (centrifugation at 4000 rpm, for 10 minutes) and the supernatant is analyzed.
(182) In order to follow the fermentation, the wort is removed, filtered through kieselguhr, and then a percentage measurement in mass of dry extract of the wort is carried out. This measurement is expressed in degrees Plato (noted °Plato) and implemented by means of an automatic densitometer Anton Paar DMA 35. A monitoring of the yeast population is also carried out.
(183) Results
(184) Fermentation is accelerated with barley malt extract. At the same time, on D0+3, less fermentable sugars remain in the presence of barley malt than during the fermentation of the control consisting of barley malt alone denatured by heat, negative control and control positive with Brewers Clarex® (
(185) This application includes a Sequence Listing associated with its image file at the United States Patent Office as follows:
(186) Name of file: P11814US00_NAT_STAGE_SEQUENCE_LISTING
(187) Date of Creation: Oct. 27, 2017
(188) Size of the File in Bytes: 21,666