WO2016004488A1 - Método para detecção de alfa-amilase em açúcares ou intermediários - Google Patents

Método para detecção de alfa-amilase em açúcares ou intermediários Download PDF

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Publication number
WO2016004488A1
WO2016004488A1 PCT/BR2014/000228 BR2014000228W WO2016004488A1 WO 2016004488 A1 WO2016004488 A1 WO 2016004488A1 BR 2014000228 W BR2014000228 W BR 2014000228W WO 2016004488 A1 WO2016004488 A1 WO 2016004488A1
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WO
WIPO (PCT)
Prior art keywords
sugar
amylase
alpha
starch
sample
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PCT/BR2014/000228
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English (en)
French (fr)
Portuguese (pt)
Inventor
Jadyr Mendes de OLIVEIRA
Maurício Sergio ESTELLER
Rafael De Araújo BORGES
Rafael Ferraz ALVES
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Prozyn Indústria E Comércio Ltda
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Priority to BR112016021309A priority Critical patent/BR112016021309B8/pt
Priority to PCT/BR2014/000228 priority patent/WO2016004488A1/pt
Publication of WO2016004488A1 publication Critical patent/WO2016004488A1/pt

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
    • G01N11/02—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material
    • G01N11/04—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material through a restricted passage, e.g. tube, aperture
    • G01N11/06—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties by measuring flow of the material through a restricted passage, e.g. tube, aperture by timing the outflow of a known quantity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/10—Starch-containing substances, e.g. dough

Definitions

  • the present invention relates to a method for detecting the presence of residual alpha-amylase enzymes in raw or intermediate sugar samples wherein said method evaluates sample viscosity by Falling Number.
  • the method according to the invention utilizes the Falling Number rheology apparatus (instrumental method). This equipment, which was originally used to measure diastatic activity in wheat flour, is used herein to evaluate the presence of process alpha-amylases remaining in starch hydrolysis during sugar production.
  • Sugarcane Saccharum officinarum
  • Grasses are a large family of monocotyledonous plants, with surrounding leaves and generally hollow stem. From its inception to the present day it has been undergoing modifications, which has resulted in several species, which differ mainly in fiber and sugar content.
  • Today most of the cultivated sugar cane is a hybrid of original plant with other species of the same family.
  • Sucrose the main energy reserve of the plant is stored predominantly in the stalks of the plant.
  • Sugar cane is basically made up of a root system, stalks and leaves that are arranged around the cane. This energy molecule can also be found at the top of the plant, where the "tip” or "palm heart” is located. With more advanced sugarcane maturation, sucrose is being housed in the intercellular spaces (Batta and Singh, 1986).
  • the chemical composition of sugarcane is very variable depending on the climatic conditions, the physical, chemical and microbiological properties of the soil, the type of cultivation, the variety, the age, the ripening stage, the sanitary state, among others. factors. In general the main constituents of sugarcane are distributed as described in Table 1 (COPERSUCAR, 2007).
  • Sucrose gives foods different properties of industrial interest, such as: sweetness, increase in viscosity and consistency, preservation, substrate for alcoholic fermentation, decrease in freezing point, color and enhancement of aroma and flavor.
  • Sucrose is presented as VHP sugar, VVHP sugar, crystal sugar, sugar amorphous refined, powdered sugar, granulated refined sugar, liquid sugar, invert liquid sugar.
  • Starch is considered an undesirable substance present in sugarcane as it causes problems during processing in mills and refineries.
  • main problems related to this molecule throughout the process we can mention: the increase of the broth and molasses viscosity; difficulty of crystallization; reduced spin rate and poor sugar quality (color, black dots, filterability, etc.) (EGGLESTON et al., 2007).
  • ESGLESTON et al. 2007
  • a study by CUDDIHY et al. (2006) showed that - starch concentrations between 200-250 ppm can cause problems during refinery raw sugar processing.
  • the removal of starch in sugarcane processing becomes indispensable to increase production capacity, volume and quality (ANYNGWA et al., 1993).
  • Thermostable bacterial ⁇ -amylase is used throughout the harvest in sugar mills.
  • the main function of this molecule is to hydrolyze the ⁇ -1,4 glycosidic bonds of starch throughout the industrial process (VALLEE et al., 1959), thereby facilitating the filtration and crystallization of sucrose.
  • VALLEE et al., 1959 the industrial process
  • high starch concentrations in sugarcane juice interfere with processing, increasing viscosity, decreasing filtration rate and reducing raw sugar yield.
  • Thermosistant alpha-amylases act on the decanter under the extreme conditions of this step (105 ° C for 1.5 hours). However, due to the high thermotolerance of this enzyme, it remains active in sugar after all subsequent steps, resulting in residual alpha-amylase activity in the final product.
  • thermo-resistant enzymes in the evaporator (65-75 ° C for 30-90 minutes) and does not allow optimal conditions for enzyme action does not effectively contribute to its inactivation, also resulting in residual enzymatic activity in the final product. Even intermediate thermostability enzymes dosed at this point do not undergo significant destabilization, leaving residual activity for the following steps. From the evaporation stage, the temperature of the next process unit processes tends to decrease, which allows the alpha-amylases to remain active in the subsequent process steps. Under these conditions, the sugar produced may carry alpha-amylase activity, which is extremely deleterious for some foods that employ sugar and starch in their composition.
  • Iodometric Method This method is based on the discoloration of the starch-iodine complex due to the action of the enzymes present in the solution to be tested. Thus, it is possible to correlate the amount of enzyme present with the absorbance of the solution containing the starch-iodine complex. It is noteworthy the need for a standard alpha-amylase curve in order to estimate the amount of enzymes. However, this method was not efficient nor viable for the determination of alpha-amylases in raw sugar, since it is not possible to establish a linear correlation between enzyme concentration and enzyme activity units. Another disadvantage of the method is the low sensitivity. Thus, although well known primarily for the determination of alpha-amylases in the baking industry, this method is not suitable for quantifying residual alpha-amylases in sugar samples.
  • Phadebas Method This method is based on the hydrolysis of insoluble starch-cibacron blue polymer by ⁇ -amylases and the release of blue-colored fragments that can be quantified by absorbing at 620 nm wavelength. EGGLESTON (2005) reports that this method is more suitable for the qualitative determination of ⁇ -amylases in raw sugar.
  • the main disadvantages of this methodology is the high cost of the Phadebas reagent and the specific filter paper (W atman) required to perform the assay. Also, the reagent shelf life is quite short. ?
  • Falling Number (FN) equipment is commonly used by the milling industry to define the amount of alpha-amylases in wheat (or flour) blends to correlate this parameter with the quality of the sample.
  • FN Falling Number
  • To perform this test in the FN requires an amount of 10 grams of flour sample to be tested, plus 25 mL of deionized water.
  • Flour is first added to the glass viscometric tubes, followed by the addition of water. Immediately thereafter the tubes are shaken in homogenizing equipment and analyzed according to the drop time (TQ). Briefly, the correlation of fall time with flour quality is as follows:
  • the present invention proposes a novel method for detecting alpha-amylase residual in raw or intermediate sugar samples and other process materials (primary broth, secondary broth, final honey, etc.) using the method of Falling Number originally used to measure diastatic activity in wheat flour.
  • This methodology has as its main advantages the speed and ease of execution, which can be performed by a trained technician and the result obtained in less than 5 minutes.
  • the main expense would be the purchase of equipment and routine maintenance. Costs for reagents would be minimal, as the main compounds needed would be: starch (from various sources) and distilled water for dilution of the sugar sample.
  • the present invention relates to a method for detecting alpha-amylase in sugars or intermediates wherein said method evaluates sample viscosity by Falling Number and comprises the steps of:
  • step (b) adding the liquid sugar sample or intermediates obtained in step (a) to an appropriate container containing one or more starch sources;
  • step (c) heating the solution obtained in step (c) at the same time as said solution is stirred with a rod until gelatinization;
  • step (f) To compare the drop time obtained in step (f) with the drop time of a sugar sample without alpha-amylase (control sample).
  • the present invention proposes a rapid innovative method of detecting residual alpha-amylase activity in sugar and / or intermediates of the sugar production process.
  • the method is based on the principle of the rheological apparatus for determining wheat flour diastatic power, the Falling Number (FN).
  • the present invention is a method for detecting alpha-amylase in sugars or intermediates wherein said method evaluates sample viscosity by Falling Number and comprises the steps of:
  • step (b) adding the liquid sugar sample or intermediates obtained in step (a) to an appropriate container containing one or more starch sources;
  • step (c) heating the solution obtained in step (c) at the same time as said solution is stirred with a rod until gelatinization;
  • step (f) To compare the drop time obtained in step (f) with the drop time of a sugar sample without alpha-amylase (control sample).
  • sugar is defined as sucrose crystals (disaccharides) obtained by processing sugar cane within the Plants used for various applications, especially in food; and its intermediates are defined as the products obtained after various stages of sugar production such as juice (between milling and decanter), syrup (syrup tank), honey A, honey B, rich honey, poor honey, final honey, molasses or mixtures thereof (residue obtained after the syrup centrifugation).
  • the liquid sugar sample or intermediates is prepared from solid (raw) sugar mixed with distilled water, the solid sugar being chosen from raw, demerara, brown sugar, VHP, VVHP , white sugar, white crystal (types I, II, III, IV and Special), refined sugar or mixtures thereof.
  • solid state sugar its concentration varies between 50 and 600 g / L; preferably between 80 g / l and 550 g / l; and more specifically between 110 g / L and 500 g / L.
  • prepared solid state sugar samples are employed at a concentration of 500 g / l.
  • the liquid sugar sample or intermediates may be prepared from liquid sugar chosen from cane juice, primary juice, secondary juice, mixed juice, clarified juice, broth juice, sulphite juice, recovered juice, syrup, honey. , honey B, rich honey, poor honey, final honey, molasses or mixtures thereof.
  • the brix solids content ranges from 5 to 60 ° Brix; preferably between 8 and 55 ° Brix, more specifically between 11 and 50 ° Brix.
  • the solids content is 20 ° Brix.
  • starch source employed in the method according to the invention wheat flour, iron-fortified and / or folic acid enriched wheat flour, rice flour, wheat bran, cornmeal, cornmeal, cassava, tapioca, manioc starch, oatmeal, rye flour, barley flour, soya flour, sunflower flour, soluble starch, potato starch, modified starch or mixtures thereof.
  • modified starch is employed.
  • the amount of starch source used varies between 1.5 g and 10 g; preferably between 2 g and 8 g; more preferably, between 3 g and 6 g, depending on the starch content of the source.
  • the starch content contained in the container to which the liquid sugar sample of step (a) will be added ranges from 0.5 g to 4 g; preferably between 1 g and 3.8 g.
  • the viscosity of the solution obtained in step (d) (considering the starch in gelatinized form) must be within the range of the device which can vary between 400 and 600 cP, preferably between 400 and 500 cP.
  • Viscosity verification was performed on the Viscolite VL700-T15 (small-sample portable viscometer) equipment. Note that the viscosity values may vary without significant changes depending on the equipment.
  • the heating temperature ranges between 60 ° C and 100 ° C; preferably between 95 ° C and 100 ° C.
  • bivalent calcium ions as an enzyme inducer are added in step (b) to the liquid sugar sample at a concentration ranging from 30 to 100 ppm relative to starch content; preferably 50 to 60 ppm.
  • a more concentrated solution can be worked on, always paying attention to the viscosity of the final solution in step (d), which must be within the detection limit of the apparatus.
  • control an enzyme-free raw sugar sample (control) in order to compare the sample to be analyzed according to the time of drop. If a control sugar is not possible, an endo / exo protease (hydrolysis at -50 ° C for 30 minutes) that will hydrolyze any protein present (alpha-amylase) can be used and used as a white solution.
  • FIGURE 1 illustrates the method developed for detecting residual enzymes in sugar, wherein A: starch source; B: liquid sugar sample; C: Falling Number equipment; D: Analysis of the viscosity of solution A + B. The method is based on the viscosity of a starch solution interacting with a sugar solution.
  • modified starch (Amidomax 5550-Cargill) and a sugar solution to be tested at a concentration of 500 g / L were used. The test was conducted in a temperature controlled room between 22 ° C and 24 ° C for 3 hours.
  • FIGURE 2 presents the results obtained for the control sample (without enzyme use) and the sample using the thermoresistant alpha-amylases.
  • This modified Bernfeld method by BOURNE et al. (1979), is based on the measurement of reducing sugars formed from the soluble starch substrate.
  • substrate preparation the suspension of 3 g of soluble starch (Merck) in 50 ml of distilled water was heated for 10 minutes by boiling. After cooling to room temperature 50 mL of 0.2 M phosphate buffer pH 6.9 was added and the volume was made up to 100 mL with distilled water.
  • modified starch (Amidomax 5550-Cargill) and a sugar solution to be tested at a concentration of 500 g / L were used. The test was conducted in a temperature controlled room between 22 ° C and 24 ° C for 3 hours.
  • FIGURE 4 presents the results obtained for the control sample (without enzyme use) and the sample using the heat-resistant amylases applied to the evaporator.
  • FIGURE 5 presents the result obtained by the modified Bernfeld method which again reveals that sugar samples obtained without alpha-amylases showed no evidence of enzyme residues.
  • undamaged sugars were dosed with alpha-amylases at a concentration of 8 ppm in the evaporator with enzyme concentration values close to 1 ppm in the final product.
  • the results corroborate those obtained according to the methodology based on viscosity / drop time.
  • sucrose metabolism in sugar cane grown under varying climatic conditions: synthesis and storage of sucrose in relation to the activities of sucrose synthase, sucrose-phosphate synthase and invertase. Phytochemistry, v. 25, noll, p.2431-2437, 1986.
  • FIGUEIRA J.A. - Determination and characterization of starch in sugarcane and adequacy of methodology for determination of ⁇ -amylase in raw sugar. Master's thesis. Unicamp, Campinas, 2009. 93p.
  • HAGBERG S. A rapid method for determining alpha-amlylase activity. Cereal Chem. 37: 218-212, 1960.
  • KAMPEN W.H. - The action of alpha-amylase on sugarcane starch, www.midlandresearchlabsinc.com;
  • MOHABIR MOHABIR, R; KHES A, Y. - A study of enzyme activity and starch reduction, Tongaat-Hulett Sugar Limited TMD, no. 49/03, 27 october, 2003, 14 pp.
  • VALLEE BL; STEIN, EA; SUMMERWILL, WN; FISHER EH - Metal content of amylases of various origins. Journal Biology Chemistry, v. 234, p. 2901-2905, 1959.
  • RAVAGNANI EM, Borges R., Esteller MS - Alpha Amylases and Dextranases. Milk and derivatives. No. 130, December 2011.

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PCT/BR2014/000228 2014-07-11 2014-07-11 Método para detecção de alfa-amilase em açúcares ou intermediários WO2016004488A1 (pt)

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BR112016021309A BR112016021309B8 (pt) 2014-07-11 2014-07-11 Método para detecção de alfa-amilase em açúcares ou intermediários
PCT/BR2014/000228 WO2016004488A1 (pt) 2014-07-11 2014-07-11 Método para detecção de alfa-amilase em açúcares ou intermediários

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1215347A (en) * 1968-12-02 1970-12-09 Falling Number Ab An apparatus for measuring consistency or viscosity
GB1327134A (en) * 1970-10-26 1973-08-15 Ogilvie Flour Mills Co Ltd Heat-treated flour process for making it and products containing it
JPH065699A (ja) * 1992-06-22 1994-01-14 Kyocera Corp 画像装置

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1215347A (en) * 1968-12-02 1970-12-09 Falling Number Ab An apparatus for measuring consistency or viscosity
GB1327134A (en) * 1970-10-26 1973-08-15 Ogilvie Flour Mills Co Ltd Heat-treated flour process for making it and products containing it
JPH065699A (ja) * 1992-06-22 1994-01-14 Kyocera Corp 画像装置

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CURIC, D.; ET AL.: "The influence offungai cx- amylase supplementation on amylolytic activity and baking quality of flour.", INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY, vol. 37, 2002, pages 673 - 680 *
FIGUEIRA, J. ET AL.: "Determinação e caracterização de amido em cana-de-açúcar e adequação de metodologia para determinação de a-amilase em açúcar bruto.", DISSERTAÇÃO (MESTRE EM CIÊNCIA DE ALIMENTOS, 2009, pages 93 *
PERTEN, H.: "A Modified Falling-Number Method Suitable for Measuring Both Cereal and Fungal Alpha-Amylase Activity.", CEREAL CHEMISTRY, vol. 61, no. 2, 1984, pages 108 - 111 *
RE, R. ET AL.: "da. Estudos sobre a determinacão da atividade alfa- amilase em farinha de trigo através de método viscosimétrico.", 1989, pages 120 *
ZENEBON, O.; ET AL., MÉTODOS. FISICO-QUIMICOS PARA ANÁLISE DE ALIMENTOS., 2008, São Paulo, pages 1020 *

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BR112016021309A2 (pt) 2017-08-15
BR112016021309B8 (pt) 2021-09-08

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