METHOD FOR PREPARING FLOUR-BASED STEAMED PRODUCTS BY USING THERMOSTABLE GLUCOAMYLASSE - Patent application

Abstract

The present invention relates to a method for producing a flour-based steamed product, the method comprising providing flour and a dough or paste comprising a thermostable glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and steaming the dough or paste to produce a flour-based steamed product, as well as a composition comprising said thermostable glucoamylase.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to an enzymatic process for preparing flour-based steamed products, in particular a process for preparing flour-based steamed products such as steamed bread with a thermostable glucoamylase. [Background technology]

[0003] Flour-based steamed products, such as steamed bread, are one of the traditional staple foods for the Chinese, especially for the Chinese in North China. In recent years, with the development of industrialization of the food industry, the industrialization of the production of flour-based steamed products has gradually increased.

[0004] WO 2011 / 039324 disclosed a method for preparing steamed bread, comprising the steps of preparing a dough used to prepare the steamed bread with one or more maltogenic alpha-amylases, one or more raw amylolytic enzymes and at least one lipolytic enzyme, wherein the enzymatic process retards staling of the steamed bread product.

[0005] In daily life, we often buy / prepare a lot of steamed bread at one time and eat it in several portions. The prepared steamed bread is often stored at room temperature, low temperature or frozen for some time, and before eating, it is necessary to re-steam the steamed bread again to restore its softness and mouthfeel. However, re-steaming can adversely affect the crumb structure, external color, chewiness and / or elasticity of the flour-based steamed products. Summary of the Invention [Problem to be solved by the invention]

[0006] However, as such, some progress has been made in extending the shelf life of steamed bread using enzyme treatment. However, there is still a need to provide new enzymatic solutions with improved performance in preserving freshness or preventing spoilage of steamed flour-based products, even if such steamed flour-based products need to be re-steamed. [Means for solving the problem]

[0007] The inventors have discovered that the thermostable glucoamylase of the present invention exhibits greatly improved performance in preserving the freshness or preventing deterioration of flour-based steamed products, such as steamed bread, prepared by a dough steaming step.

[0008] Another improved performance of the thermostable glucoamylase of the present invention was that the thermostable glucoamylase of the present invention surprisingly and significantly improved the quality of flour-based steamed products after resteaming, such as improved appearance whiteness, softness, springiness, crumb structure and / or cohesiveness of the resteamed products.

[0009] Another improved performance of the thermostable glucoamylase of the present invention was that it increased the sweetness or sweetness of the product, whose natural sweetness is a pleasant taste, which further allowed for a reduction in the amount of sugar added in traditional recipes.

[0010] Thus, in a first aspect, there is provided a method for producing a flour-based steamed product, comprising the steps of: a) providing a dough or paste comprising flour and a glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; and b) A process which comprises steaming a dough or paste to produce a flour-based steamed product.

[0011] In a preferred embodiment of the invention, the glucoamylase is a mature thermostable variant of a parent glucoamylase.

[0012] A second aspect of the invention relates to a dough premix or paste premix comprising a dough or paste and a mature thermostable variant of a parent glucoamylase as defined in the present invention.

[0013] In a preferred embodiment of the present invention, the flour-based steamed product of the present invention has reduced hardness and / or improved elasticity and thus improved storage stability compared to conditions other than no enzyme addition to the dough / paste, where all other conditions are the same.

[0014] In a preferred embodiment of the present invention, the flour-based steamed product of the present invention has improved sensory evaluation after storage at room temperature or low temperature, preferably for some time, compared to conditions where all other conditions are the same except that no enzyme is added to the dough / paste.

[0015] In a preferred embodiment of the present invention, the resteamed flour-based steamed product has improved sensory evaluation compared to conditions other than no enzyme addition to the dough / paste, where all other conditions are the same.

[0016] In a preferred embodiment of the present invention, the sensory evaluation is preferably an overall evaluation, an average value of softness, springiness, surface whiteness, crumb structure, moisture, cohesiveness, chewiness and / or sweetness of the flour-based steamed product.

[0017] Preferably, the glucoamylase of the present invention is at least 71% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, such as at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, more preferably the glucoamylase is a mature thermostable variant of a parent glucoamylase. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 shows a multiple alignment of the amino acid sequences of the following mature proteins: - wild type AMG (PoAMG) from Penicillium oxalicum according to SEQ ID NO: 1 - PoAMG variant designated "AMG NL" according to SEQ ID NO: 2 - PoAMG variant designated "AMG anPAV498" according to SEQ ID NO: 3 - PoAMG variant designated "AMG JPO001" according to SEQ ID NO: 4 - PoAMG variant designated "AMG JPO124" according to SEQ ID NO: 5 - PoAMG variant designated "AMG JPO172" according to SEQ ID NO: 6 - wild type AMG (PoAMG) from Penicillium miczynskii according to SEQ ID NO: 7 - wild type AMG (PoAMG) from Penicillium russellii according to SEQ ID NO: 8 - PoAMG variant designated "AMG JPO172" according to SEQ ID NO: 9 Wild-type AMG (PoAMG) derived from P. glabrum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] definition In accordance with this Detailed Description, the following definitions apply: Please note that the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0020] Throughout this specification, values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. For example, the range "about 0.1 percent to about 5 percent" or "about 0.1 percent to 5 percent" should be interpreted not only to include about 0.1 percent to about 5 percent, but also to include the individual values ​​(e.g., 1 percent, 2 percent, 3 percent, and 4 percent) and subranges (e.g., 0.1 percent to 0.5 percent, 1.1 percent to 2.2 percent, 3.3 percent to 4.4 percent) within the recited range. Unless otherwise indicated, the description "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the description "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z".

[0021] Unless otherwise defined or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0022] Glucoamylase: The term glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is defined as an enzyme that catalyzes the release of D-glucose from the non-reducing end of starch or related oligosaccharide and polysaccharide molecules. Glucoamylase is also called amyloglucosidase and glucan 1,4-alpha-glucosidase (EC 3.2.1.3), and more commonly, they are referred to as AMG. A glucoamylase unit (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromole of maltose per minute under standard conditions: 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate 0.1 M, reaction time 5 minutes.

[0023] Parent or parent glucoamylase: The term "parent" glucoamylase, as used herein, refers to a glucoamylase that is modified to produce a variant glucoamylase of the invention. This term also refers to a polypeptide to which the variant of the invention is compared. A parent may be a naturally occurring (wild type) polypeptide or even a variant thereof prepared by any suitable means. By way of example, a parent protein may be a variant of a naturally occurring polypeptide in which the amino acid sequence has been modified or altered. As such, a parent glucoamylase may have one or more (or one or several) amino acid substitutions, deletions and / or insertions. As such, a parent glucoamylase may be a variant of a parent glucoamylase. A parent may also be an allelic variant, which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus.

[0024] Mature Polypeptide: The term "mature polypeptide" is defined herein as a biologically active polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. A mature polypeptide sequence lacks a signal sequence, which may be determined using techniques known in the art (see, e.g., Zhang and Henzel, 2004, Protein Science 13:2819-2824). The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide.

[0025] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0026] For the purposes of the present invention, sequence identity between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (identical residues × 100) / (length of alignment – ​​total number of gaps in alignment)

[0027] Variant: The term "variant" refers to a polypeptide having glucoamylase activity that comprises an alteration, i.e. a substitution, insertion and / or deletion, at one or more (e.g. several) positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid, a deletion refers to the removal of an amino acid occupying a position, and an insertion refers to the addition of an amino acid adjacent to and immediately following the amino acid occupying a position. A variant of the invention has at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the glucoamylase activity of the polypeptide of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0028] Sensory evaluation: To perform a sensory evaluation, a panel (e.g., at least three well-trained people) is used to judge the quality of the steamed products.

[0029] Improved softness of steamed products: The term "improved softness of steamed products" is the opposite of "firmness / hardness" and is defined herein as the property of a steamed product that is more easily compressed compared to a control, assessed empirically by trained human hand or mouth or measured by, for example, a texture analyzer known in the art (e.g., TAXT2 or TA-XT Plus manufactured by Stable Micro Systems Ltd, Surrey, UK).

[0030] Improved springiness of steamed products: The term "improved springiness of steamed products" is defined herein as the property of a steamed product which recovers more easily after being pressed compared to a control, as assessed empirically by trained human hand or mouth or measured by, for example, a texture analyzer known in the art (e.g., TAXT2 or TA-XT Plus manufactured by Stable Micro Systems Ltd, Surrey, UK).

[0031] Improved appearance whiteness of steamed products: The term "improved appearance whiteness of steamed products" is defined herein as the property of a steamed product having a whiter appearance on the surface compared to a control, typically as assessed visually by a well-trained person.

[0032] Improved crumb structure of steamed products: The term "improved crumb structure of steamed products" is defined herein as the property of a steamed product having finer air bubbles and / or thinner cell walls in the crumb and / or a more uniform / homogeneous distribution of air bubbles in the crumb compared to a control, typically as assessed visually by a well-trained individual.

[0033] Improved moisture in steamed products: The term "improved moisture in steamed products" is defined herein as the property of a steamed product that feels cooler to the touch or has a cooler mouthfeel compared to a control, and is typically assessed empirically by a well-trained individual.

[0034] Improved cohesiveness of steamed products: The term "improved cohesiveness of steamed products" is defined herein as the property of a steamed product that exhibits less crumb collapse compared to a control, and is typically assessed empirically by a well-trained person.

[0035] Improved chewiness of steamed products: The term "improved chewiness of steamed products" is defined herein as the property of a steamed product that involves more chewing effort before swallowing compared to a control, typically as assessed empirically by a skilled baker.

[0036] Improved sweetness of steamed products: The term "improved sweetness of steamed products" is defined herein as the property of a steamed product that has a sweeter taste compared to a control, typically as assessed by a well-trained person.

[0037] Thermostability Improved: Thermostability Improved (Td) in °C is a measure of how much the variants have improved thermostability over their parent glucoamylase under the same conditions, determined as exemplified herein.

[0038] nomenclature For the purposes of the present invention, the nomenclature [Y / F] means that the amino acid at this position may be tyrosine (Try, Y) or phenylalanine (Phe, F). Similarly, the nomenclature [V / G / A / I] means that the amino acid at this position may be valine (Val, V), glycine (Gly, G), alanine (Ala, A) or isoleucine (Ile, I), as well as for other combinations described hereinafter. The amino acid X is defined to be any of the 20 naturally occurring amino acids, unless otherwise specified.

[0039] Rules for Naming Variants For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid residue in another glucoamylase. As such, all referenced positions and specific substitutions refer to the numbering used in SEQ ID NO: 1. However, the skilled artisan will recognize that the sequence of any other sequence disclosed herein may also be used to determine the corresponding amino acid residue in another glucoamylase polypeptide. The amino acid sequence of another glucoamylase is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16; 276-277), preferably version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5 and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0040] For proteins of known structure, several tools and resources are available for searching and generating structural alignments. For example, structures of proteins of the SCOP superfamily have been aligned and the alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), and implementations of these algorithms can be further used to query structural databases with structures of interest to find potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0041] In describing the variants of the present invention, the following nomenclature is adopted for ease of reference: accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0042] Substitutions. In the case of amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine with alanine at position 226 is designated "Thr226Ala" or "T226A". Multiple mutations are separated by additional marks ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of serine (S) with glycine (G) and phenylalanine (F) with arginine (R), respectively, at positions 205 and 411.

[0043] Deletions. In case of amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of glycine at position 195 is designated as "Gly195*" or "G195*". Multiple deletions are separated by additional marks ("+"), e.g., "Gly195*+Ser411*" or "G195*+S411*".

[0044] Insertions. In the case of amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of a lysine after a glycine at position 195 is designated as "Gly195GlyLys" or "G195GK". An insertion of multiple amino acids is designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, an insertion of a lysine and an alanine after a glycine at position 195 is designated as "Gly195GlyLysAla" or "G195GKA".

[0045] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. In the above example, the sequence would thus be:

[0046] [Table 1]

[0047] Multiple modifications. Variants containing multiple modifications are separated by additional signs "+", for example "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent the substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

[0048] Different modifications. When different modifications can be introduced at a position, the different modifications are separated by a comma, for example "Arg170Tyr,Glu" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly" and "Tyr167Ala+Arg170Ala".

[0049] Detailed Description of the Invention A first aspect is a method for producing a flour-based steamed product, comprising the steps of: a) providing a dough or paste comprising flour and a glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; and b) steaming a dough or paste to produce a flour-based steamed product.

[0050] In a preferred embodiment of the invention, the glucoamylase is a mature thermostable variant of a parent glucoamylase.

[0051] A second aspect of the invention relates to a dough premix or paste premix comprising a dough or paste and a mature thermostable variant of a parent glucoamylase as defined in the present invention.

[0052] In a preferred embodiment of the present invention, the flour-based steamed product of the present invention has reduced hardness and / or improved elasticity and thus improved storage stability after storage at room temperature or at a low temperature of 4°C for 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6 or 7 days or after storage at -20°C for up to 1, 2, 3, 4, 5, 6 months, compared to conditions where other conditions are the same except that no enzyme is added to the dough / paste.

[0053] In a preferred embodiment of the present invention, the flour-based steamed product of the present invention has improved sensory evaluation after storage at room temperature or at a temperature as low as 4°C for 0.1, 0,25, 0.5, 1, 2, 3, 4, 5, 6 or 7 days or after storage at -20°C for up to 1, 2, 3, 4, 5, 6 months compared to conditions where all other conditions are the same except no enzyme is added to the dough / paste.

[0054] In a preferred embodiment of the present invention, when the flour-based steamed product of the present invention is re-steamed after storage at room temperature or at a low temperature of 4°C for 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6 or 7 days or after storage at -20°C for up to 1, 2, 3, 4, 5, 6 months, compared to conditions where all other conditions are the same except no enzyme is added to the dough / paste, the re-steamed product has an improved sensory evaluation.

[0055] In a preferred embodiment of the present invention, the sensory evaluation is preferably an overall evaluation, an average value of softness, springiness, whiteness of appearance, crumb structure, moisture, cohesiveness, chewiness and / or sweetness of the flour-based steamed product.

[0056] In another preferred embodiment of the present invention, the flour-based steamed product has at least the same sweetness or sweetness as a control product made with twice the amount of mature glucoamylase, the amino acid sequence of which is shown in SEQ ID NO:10.

[0057] Another aspect is a method for producing a flour-based boiled product, comprising the steps of: a) providing a dough comprising flour and a glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8; and b) boiling dough to produce flour-based boiled products The present invention relates to a method comprising the steps of:

[0058] In a preferred embodiment of the invention, the glucoamylase is a mature thermostable variant of a parent glucoamylase.

[0059] Flour-based steamed products: As used herein, "flour-based steamed product" refers to any flour-based product prepared by steaming into a dough or paste. Examples of flour-based steamed products include steamed bread, such as North China steamed bread and South China steamed bread, steamed stuffed steamed bread (baozi), steamed twisted roll (huajuan), steamed roll (juan zi), steamed dumpling, steamed noodles (men mian), Chinese New Year cake (nian gao), steamed sponge cake (fa gao) or steamed sponge rice cake (mi fa gao). The flour-based steamed product may contain one or more additional ingredients, such as meat (e.g. pork, beef, chicken or fish), vegetables (e.g. mushrooms, broccoli and other green vegetables), fruits (e.g. dates and jujubes), candy, cheese and milk (or other dairy products) and combinations thereof.

[0060] The present invention relates to a dough or paste comprising the thermostable glucoamylase of the present invention.As used herein, "dough" refers to any dough used to prepare a flour-based steamed product, such as steamed bread.The dough used to prepare a flour-based steamed product may be made from any suitable flour raw material, such as flour from cereal sources, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour or sorghum flour, potato flour, and combinations thereof (e.g., wheat flour combined with one of the other flour raw materials; rice flour combined with one of the other flour raw materials).

[0061] The dough of the present invention is usually a leavened or fermented dough. The dough can be leavened in various ways, such as by adding a dough ingredient such as a chemical leavening agent (e.g. sodium bicarbonate) or by adding a leavening agent (fermented dough), but it is preferred to leaven the dough by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae.

[0062] The present invention relates to flour premixes comprising the thermostable glucoamylase of the present invention, which may comprise any suitable flour ingredient, such as flour from a cereal source, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour or sorghum flour, potato flour and combinations thereof (e.g. wheat flour combined with one of the other flour ingredients; rice flour combined with one of the other flour ingredients).

[0063] Methods for preparing steamed bread are well known in the art and include, for example, the "straight dough process" and the "sponge and dough process", non-limiting examples of which are provided in the "Materials and Methods" section below. The process for preparing steamed bread generally involves the successive steps of making dough (with an optional fermentation step), sheeting, shaping, fermenting and then steaming the dough, which steps are well known in the art. If an optional fermentation step is used, preferably more flour is added, and alkali may be added during the second fermentation step to neutralize the acid that has been or will be produced.

[0064] Methods for preparing steamed sponge cake are well known in the art, including, for example, the method for preparing steamed sponge cake, comprising preparing a paste from a flour-containing material, fermenting and steaming to produce a steamed sponge cake.Preferably, the paste is rice paste.Preferably, the flour can be of cereal origin, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour or sorghum flour, potato flour and combinations thereof.

[0065] Preferably, the finished cake has a fine mesh structure, soft taste, pleasant wine aroma and lactic acid flavor produced by fermentation. Preferably, the paste contains the thermostable glucoamylase of the present invention. Preferably, the steamed sponge cake is made from rice, for example, the method includes soaking rice in water, grinding the soaked rice and sieving to obtain rice paste or making rice paste by adding water to steamed sponge cake premix powder, fermenting the rice paste at 30-40°C for about 1-25 hours, then adding sucrose and baking powder, mixing the mixture uniformly, and steaming for about 15 minutes to make the steamed sponge cake. The paste is generally fermented by the addition of a suitable yeast culture, for example a culture of Saccharomyces cerevisiae (baker's yeast) or a chemical leavening agent, as is well known in the art.

[0066] In addition to preparing fresh steamed bread dough or steamed bread products, the present invention relates to a method for preparing frozen steamed bread dough, which may be advantageous for storage and / or sale. An example of a method for preparing frozen steamed bread dough includes the steps of making a dough (with an optional fermentation step), sheeting, shaping, fermenting, and freezing the dough. The present invention also relates to a frozen steamed bread dough comprising the thermostable glucoamylase of the present invention.

[0067] The present invention is particularly useful for preparing steamed bread dough and steamed bread products in industrial processes, i.e., processes in which the dough used to prepare the steamed bread and / or steamed bread products is prepared mechanically using automated or semi-automated equipment. The present invention provides a significant advantage in that the steamed bread can be quickly prepared using an automated or semi-automated process, and the steamed bread can be stored for sale and consumption by consumers, substantially maintaining the quality of steamed bread freshly prepared on the same day, even more than 24 hours after preparation.

[0068] The process of preparing steamed bread generally includes the successive steps of making dough (and optional fermentation), sheeting, shaping, fermentation, steaming and packaging. If an optional fermentation step is used, more flour is preferably added, and during the second fermentation step, alkali may be added to neutralize the acid produced or to be produced. In the industrial steamed bread production process according to the present invention, one or more of these steps, such as sheeting, shaping, fermentation, steaming and / or packaging, are carried out using automated or semi-automated equipment.

[0069] In one embodiment, the glucoamylase according to the invention may be added to the flour or dough or paste in an amount of 0.01-1,000 mg enzyme protein (mg EP) per kg of flour, preferably in an amount of 0.01-500 mg enzyme protein (mg EP) per kg of flour, even more preferably in an amount of 0.1-100 mg enzyme protein (mg EP) per kg of flour.

[0070] Flour-based boiled products: As used herein, "flour-based boiled product" refers to any flour-based product prepared by boiling dough. Examples of flour-based boiled products include traditional Chinese staples such as noodles, dumplings and wontons, rice dumplings and rice noodles, and dried products such as dried and semi-dried noodles, hot dried noodles, dried and semi-dried rice noodles, and fresh rice noodles. The flour may be made from any suitable flour raw material, such as flour from cereal sources, such as rice flour, wheat flour, buckwheat flour, purple rice flour, corn flour, rye flour, barley flour, oat flour or sorghum flour, potato flour and combinations thereof (e.g. wheat flour combined with one of the other flour raw materials; rice flour combined with one of the other flour raw materials). It can be a product with or without a filling. The filling can be meat, vegetable, bean or a combination thereof. The dough of this type of product is usually not leavened by yeast. The resulting dough, with or without filling, can be processed through a variety of processes into products of various shapes.

[0071] Flour-based boiled products are usually boiled for an appropriate time depending on the thickness or filling of the product and whether it is frozen or dried before consumption. Various products are boiled in boiling water for 3 to 20 minutes to achieve sufficient gelatinization.

[0072] In one embodiment, the glucoamylase according to the invention may be added to the flour or dough in an amount of 0.01-1,000 mg enzyme protein (mg EP) per kg of flour, preferably in an amount of 0.01-500 mg enzyme protein (mg EP) per kg of flour, and even more preferably in an amount of 0.1-100 mg enzyme protein (mg EP) per kg of flour.

[0073] Glucoamylase According to the present invention, various types of glucoamylases may be used as parents for the generation of thermostable glucoamylase variants, for example, the glucoamylase may be a polypeptide encoded by a DNA sequence found in a fungal strain of the genus Aspergillus, Rhizopusor, Talaromyces or Penicillium, preferably a DNA sequence found in a fungal strain of the genus Penicillium, even more preferably a DNA sequence found in a fungal strain of Penicillium oxysporum, Penicillium oxalicum, Penicillium miczynskii, Penicillium russellii or Penicillium glabrum. Other examples of suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii.

[0074] The parent glucoamylase may be obtained from any genus of microorganism. For purposes of the present invention, the term "obtained from," when used herein in connection with a given source, is intended to mean that the parent encoded by the polynucleotide is produced by the source or strain into which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

[0075] In one aspect of the invention, the glucoamylase or parent glucoamylase may be obtained from the genus Penicillium, such as, for example, Penicillium oxalicum, Penicillium glabrum, Penicillium brasilianum, Penicillium russellii, or Penicillium miczynskii.

[0076] In one aspect, the parent fungal glucoamylase can be a Penicillium glucoamylase, such as Penicillium oxalicum glucoamylase, Penicillium glabrum glucoamylase, Penicillium brasilianum glucoamylase, Penicillium russellii glucoamylase, or Penicillium miczynskii glucoamylase.

[0077] In another embodiment, the parent glucoamylase is obtained from Penicillium oxalicum and is, for example, designated as the glucoamylase of SEQ ID NO: 1. In another embodiment, the parent glucoamylase is obtained from Penicillium oxalicum and is, for example, designated as the glucoamylase of SEQ ID NO: 6. In another embodiment, the parent glucoamylase is obtained from Penicillium oxalicum and is, for example, designated as the glucoamylase of SEQ ID NO: 7. In another embodiment, the parent glucoamylase is obtained from Penicillium oxalicum and is, for example, designated as the glucoamylase of SEQ ID NO: 8.

[0078] In one embodiment the thermostable glucoamylase of the invention is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, such as at least 71%, such as at least 72%, for example at least 73%, such as at least 74%, for example at least 75%, such as at least 76%, for example at least 77%, such as at least 78%, for example at least 79%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In one embodiment, the glucoamylase is a glucoamylase variant or a mature thermostable variant of a parent glucoamylase.

[0079] In one embodiment, the mature variant comprises at least one amino acid modification at one or more (some) or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 65, 79, 103, 132, 327, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:1.

[0080] In one embodiment, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO:1, and preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO:1.

[0081] In one embodiment, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:1, preferably the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO:1.

[0082] In one embodiment, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO:1, preferably the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:1.

[0083] In one embodiment, a mature thermostable variant has a thermostability improvement (Td) over its parent of at least 3°C, preferably at least 4°C, 5°C, 6°C, 7°C or 8°C.

[0084] In one embodiment, a mature thermostable variant has a relative activity at 91° C. of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 compared to its parent.

[0085] In one embodiment, the thermostable glucoamylase is a glucoamylase variant. In one embodiment, the glucoamylase variant of the invention has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to the polypeptide of SEQ ID NO:1 or SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 and has glucoamylase activity.

[0086] In one embodiment, the glucoamylase variant or amino acid sequence of the glucoamylase variant of the invention differs by 10 or fewer amino acids, such as 9 amino acids, 8 amino acids, 7 amino acids, 6 amino acids, 5 amino acids, 4 amino acids, 3 amino acids, 2 amino acids and 1 amino acid from SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 or from the mature polypeptide of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In one embodiment, the mature polypeptide of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8 is set forth as SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0087] In one embodiment, the amino acid sequence of the glucoamylase, glucoamylase variant, or glucoamylase variant of the invention comprises or consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0088] Amino acid changes in the variants may be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of 20-25 residues or less; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine tract, an antigenic epitope or a binding domain.

[0089] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and low molecular weight amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0090] On the other hand, the amino acid changes are of such a nature that the physicochemical properties of the polypeptide are modified, for example, the amino acid changes may improve the thermostability of the polypeptide, modify the substrate specificity, change the pH optimum, etc.

[0091] Essential amino acids within a polypeptide can be identified according to techniques known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue within the molecule to identify amino acid residues critical to the activity of the molecule, and the resulting mutant molecules are tested for glucoamylase activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of the enzyme or other biological interactions can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255;306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0092] The following shows the percent identity between the AMG amino acid sequences aligned in FIG. 1 and also provided in the sequence listing:

[0093] [Table 2]

[0094] Thermostable variants of PoAMG have been generated (see Table 2 below). In a preferred embodiment, the mature thermostable glucoamylase variant of the invention comprises one or more or all of the combinations of amino acid substitutions listed in Table 2 below.

[0095] The thermostability improvement (Td) of the variants in Table 2 are listed in Table 3, where the Td of the PoAMG variant designated "anPAV498" (parent) was set to zero. In a preferred embodiment, a mature thermostable variant of the invention has a thermostability improvement (Td) over its parent of at least 3°C, preferably at least 4°C, 5°C, 6°C, 7°C or 8°C, preferably determined as exemplified herein.

[0096] In another preferred embodiment, a mature thermostable variant of the invention has a relative activity at 91° C. of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300, compared to its parent.

[0097] Additional enzymes Optionally, one or more additional enzymes, such as alpha-amylase, maltogenic amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzymes, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetrahydrolase, glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzymes, ribonuclease, transglutaminase, and xylanase, may be used together with the glucoamylase of the present invention.

[0098] The additional enzymes may be of any origin, including mammalian, plant and microbial (bacterial, yeast or fungal) origin.

[0099] Enzyme Composition The mature thermostable glucoamylase variant of the present invention and any additional enzymes may be added to the flour or dough in any suitable form, such as for example in liquid form, particularly a stabilized liquid, or it may be added to the flour or dough as a substantially dry powder or granules.

[0100] Granules may be produced, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations may be stabilized, for example, by adding sugars or sugar alcohols or lactic acid, according to established procedures. Other enzyme stabilizers are well known in the art.

[0101] The enzymes may be added to the dough ingredients in any suitable manner, such as as individual components (separate or sequential addition of enzymes) or by adding the enzymes together in one step or in one composition.

[0102] The invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, as these embodiments are intended as illustrations of some aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention, as well as combinations of one or more of the embodiments.

[0103] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0104] Example 1: Construction of a PoAMG library The PoAMG library was constructed as follows: Forward or reverse primers were designed with 15 bp overlap with each other and with NNK or desired mutation at the target site. Inverse PCR, which results in amplification of the entire plasmid DNA sequence by the reverse primer, was carried out using a suitable template plasmid DNA (e.g., plasmid DNA containing the JPO-0001 gene) under the following conditions. The resulting PCR fragment was purified by QIAquick Gel extraction kit [QIAGEN] and then introduced into Escherichia coli ECOS Competent E. coli DH5α [NIPPON GENE CO., LTD.]. Plasmid DNA was extracted from E. coli transformants by MagExtractor Plasmid Extraction Kit [TOYOBO] and then introduced into A. niger competent cells. PCR reaction mix: PrimeSTAR Max DNA polymerase [TaKaRa] Total 25μl 1.0 μl template DNA (1 ng / μl) 9,5 μl H2O 12,5μl 2x PrimeSTAR Max Premix 1.0 μl forward primer (5 μM) 1.0 μl reverse primer (5 μM) PCR program: 98℃ / 2 minutes 25×(98℃ / 10 seconds, 60℃ / 15 seconds, 72℃ / 2 minutes) 10℃ / hold

[0105] Example 2: Screening for better thermostability The B. subtilis library constructed as in Example 1 was fermented for 3 days at 32° C. in either 96-well or 24-well MTPs containing COVE liquid medium (2.0 g / L sucrose, 2.0 g / L iso-maltose, 2.0 g / L maltose, 4.9 mg / L, 0.2 ml / L 5N NaOH, 10 ml / L COVE salts, 10 ml / L 1M acetamide). The AMG activity in the culture supernatant was then measured at several temperatures by pNPG assay as described below.

[0106] pNPG Thermostability Assay: The culture supernatant containing the desired enzyme was mixed with the same volume of pH 5.0 200 mM NaOAc buffer. 20 microliters of this mixture was dispensed into either a 96-well plate or an 8-strip PCR tube, and then heated by a thermal cycler at various temperatures for 30 minutes. The samples were mixed with 10 μl of substrate solution containing 0.1% (w / v) pNPG[wako] in pH 5.0 200 mM NaOAc buffer, and incubated at 70°C for 20 minutes for enzyme reaction. After the reaction, 60 μl of 0.1 M Borax buffer was added to stop the reaction. 80 microliters of the reaction supernatant was taken and its OD 405 The values ​​were read photometrically to assess the enzyme activity.

[0107] [Table 3]

[0108] [Table 4]

[0109] [Table 5]

[0110] [Table 6]

[0111]

Table 7

[0112]

Table 8

[0113]

Table 9

[0114]

Table 10

[0115]

Table 11

[0116]

Table 12

[0117]

Table 13

[0118]

Table 14

[0119]

Table 15

[0120]

Table 16

[0121] [Table 17]

[0122] [Table 18]

[0123] [Table 19]

[0124] [Table 20]

[0125] [Table 21]

[0126] [Table 22]

[0127] [Table 23]

[0128] [Table 24]

[0129] Example 3: Fermentation of Aspergillus niger Aspergillus niger strains were fermented in 500 ml baffled flasks containing 100 ml MU1 with 4 ml 50% urea on a rotary shaker at 220 rpm and 30° C. The culture broth was centrifuged (10,000×g, 20 min) and the supernatant was carefully decanted from the sediment.

[0130] Example 4: Purification of PoAMG (JPO-001) variants The PoAMG variants were purified by cation exchange chromatography. Each peak fraction was individually pooled and dialyzed against 20 mM sodium acetate buffer pH 5.0, and then the sample was concentrated using a centrifugal filter unit (Vivaspin Turbo 15, Sartorius). The enzyme concentration was determined by A280 value.

[0131] Example 5: Thermal Stability Determination (TSA) The purified enzyme was diluted to 0.5 mg / ml in 50 mM sodium acetate buffer, pH 5.0, and mixed with an equal volume of SYPRO Orange (Invitrogen) diluted in Milli-Q water. 18 ul of the mixture was transferred to a LightCycler 480 Multiwell Plate 384 (Roche Diagnostics) and the plate was sealed. TSA instrument parameters: Equipment: LightCycler 480 real-time PCR system (Roche Applied Science) Scanning speed: 0.02°C / sec Scanning range: 37~96℃ Integration time: 1.0 seconds Excitation wavelength: 465nm Emission wavelength: 580nm

[0132] The resulting fluorescent signals were normalized to a range of 0 and 1. Td was defined as the temperature at which the signal intensity was 0.5. The thermal stability improvements are listed in Table 3, with the Td of the PoAMG variant designated as anPAV498 being 0.

[0133] Example 6: PoAMG activity assay Maltodextrin (DE11) assay by GOD-POD method substrate solution 30g maltodextrin (pindex#2 manufactured by MATSUTANI chemical industry Co., Ltd.) 100 ml of 120 mM sodium acetate buffer, pH 5.0 Glucose CII test kit (Wako Pure Chemical Industries, Ltd.)

[0134] 20ul of enzyme sample was mixed with 100ul of substrate solution and incubated for 2 hours at set temperature. Sample was cooled on aluminum block for 3 minutes and then 10ul of reaction solution was mixed with 590ul of 1M Tris-HCl pH 8.0 to stop the reaction. 10ul of the solution was mixed with 200ul of standard solution from the test kit and then left at room temperature for 15 minutes. Absorbance was read at A505. Activity is listed in Table 3 as the relative activity of the PoAMG variant designated anPAV498.

[0135] [Table 25]

[0136] [Table 26]

[0137] [Table 27]

[0138] Example 7: Use of PoAMG in steamed bread North China steamed bread was prepared by straight dough process with recipe according to Table 4 and Table 5. All ingredients used herein were food grade, and PoAMG variant JPO-172 (75 ppm as used herein corresponds to 24.45 mg EP / 1000 g flour) and maltogenic alpha-amylase were used, where maltogenic alpha-amylase is Novamyl Boost (a product of Novozymes). Briefly, flour, yeast and steamed bread food additives were weighed and put into a dough tank (vertical mixer, DIOSNA brand), then enzyme and water were added. The mixture was mixed at low speed for 6 minutes until dough was formed and the dough surface was smooth, and the dough was sheeted until the dough reached the ideal degree. The sheeting time was dependent on the texture. After sheeting, the dough was weighed to about 110 g and molded into a steamed bread shape. The molded dough was placed in a fermentation machine at 35°C for about 40 minutes (room humidity about 80%). After fermentation, the dough was placed in a steamer (100°C) and steamed for 20 minutes. Then, the steam was turned off and after about 5 minutes, the steamed bread was taken out and cooled at room temperature for 2 hours, after which the prepared steamed bread was packaged in a sealed plastic package for texture and sensory evaluation.

[0139] Re-steaming of steamed bread: The prepared steamed bread was stored at room temperature for 24 hours or in a refrigerator at 4°C for 48 hours, and then it was re-steamed in a steamer (100°C) for about 15 minutes. Then, the steam was turned off, and about 5 minutes later, the re-steamed steamed bread was taken out for sensory evaluation.

[0140] [Table 28]

[0141] [Table 29]

[0142] Sample characterization Method for determining hardness: The steamed bread was divided by using a slicer (thickness of each steamed bread slice was 1.2 cm), and two slices of steamed bread were combined into one (thickness was 2.4 cm), and determined by TA.XT Plus texture analyzer. Using grams as the unit, the higher the hardness value, the worse the quality of the prepared steamed bread.

[0143] Method for determining springiness: The steamed bread was divided by using a slicer (thickness of each steamed bread slice was 1.2 cm). Two slices of steamed bread were combined into one slice (thickness was 2.4 cm) and determined by TA.XT Plus texture analyzer. % was used as the unit. Higher springiness value indicates better quality of prepared steamed bread.

[0144] Sensory evaluation: To perform the sensory evaluation, a panel (five well-trained people) was used to judge the quality of the steamed bread / re-steamed bread. Parameters such as softness, elasticity, whiteness of appearance, crumb structure, moisture, cohesiveness, chewiness and / or sweetness were given a score. The steamed bread prepared in batch A was given a score of 5.0 points and used as the baseline. The average value was taken for the overall evaluation, and the higher the average score, the better the quality of the steamed bread prepared.

[0145] [Table 30]

[0146] As can be seen from Table 6, when PoAMG (JPO-172) and Novamyl Boost were used during the preparation of steamed bread, compared with the control (Batch A), the hardness of the steamed bread was both significantly decreased, but the springiness of the steamed bread was also both improved. However, in comparison, PoAMG (JPO-172) showed a better effect than Novamyl Boost.

[0147] Each batch of prepared steamed bread was subjected to sensory evaluation after storage at room temperature for 24 hours, and the results are shown in Table 7. Compared with the control (Batch A), the addition of PoAMG (JPO-172) and Novamyl Boost to the dough improved the sensory evaluation of the steamed bread, such as tactile softness, tactile elasticity, and also improved the crumb structure, moisture, cohesiveness and chewiness. In addition, PoAMG (JPO-172) also improved the sweetness of the steamed bread. In contrast, PoAMG (JPO-172) was significantly better than Novamyl Boost in improving the quality of steamed bread.

[0148] [Table 31]

[0149] Each batch of prepared steamed bread was stored at room temperature for 24 hours, then re-steamed, and then cooled to room temperature, after which such re-steamed steamed bread was subjected to sensory evaluation, the results of which are shown in Table 8.

[0150] [Table 32]

[0151] Each batch of prepared steamed bread was stored at 4°C for 48 hours, then re-steamed, and then cooled them to room temperature, after which such re-steamed steamed bread was subjected to sensory evaluation, the results of which are shown in Table 9.

[0152] [Table 33]

[0153] As can be seen from Tables 8 and 9, compared with the control (Batch A), the addition of PoAMG (JPO-172) to the dough obviously improved the sensory evaluation of the resteamed steamed bread, such as softness of touch, elasticity of touch, and the crumb structure, chewiness and sweetness were all improved. PoAMG (JPO-172) also improved the whiteness of the appearance of the resteamed steamed bread. However, in contrast, even when Novamyl Boost was added, the sensory evaluation of the resteamed steamed bread was not good, and even not as good as Batch A.

[0154] Example 8: Use of POAMG in steamed sponge rice cake Steamed sponge rice cake was prepared by recipe according to Table 10 and Table 11, in brief, rice paste was prepared by mixing steamed sponge rice cake premix powder and water in a container, batch A was the control without enzyme, and PoAMG (JPO-172) was added to the rice paste as batch B. The mixture was sealed and fermented at 35°C for 16 hours, then sucrose and baking powder were added after fermentation. After that, the fermented mixture was stirred and poured into a mould and then steamed in a steamer (100°C) for about 15 minutes. Then, the steam was turned off and after about 5 minutes, the steamed sponge rice cake was taken out and cooled at room temperature for 3 hours for texture and sensory evaluation.

[0155] [Table 34]

[0156] [Table 35]

[0157] Method for determining hardness: The flat surface of the steamed sponge rice cake was cut along the outer edge of the mould (each sample had a height of 2.5 cm) and the hardness was determined by a TA.XT Plus texture analyzer. The unit used was grams, and the higher the hardness value, the worse the quality of the prepared product.

[0158] Method for determining springiness: The flat surface of the steamed sponge rice cake was cut along the outer edge of the mould (the height of each sample was 2.5 cm) and the springiness was determined by TA.XT Plus texture analyzer. The higher the springiness value, the better the quality of the prepared product.

[0159] Sensory evaluation: To carry out the sensory evaluation, a panel (five well-trained people) was used to judge the quality of the steamed sponge rice cakes. Parameters such as mouthfeel for softness, mouthfeel for moisture, mouthfeel for elasticity, crumb structure and sweetness were given scores. The steamed sponge rice cake prepared in batch A was given a score of 5.0 points and used as the baseline. The average value was taken for the overall evaluation, and the higher the average score, the better the quality of the steamed sponge cake prepared.

[0160] The sensory evaluation of the prepared steamed sponge rice cakes is shown in Table 12. When PoAMG (JPO-172) was added to the paste, the softness mouthfeel, moisture mouthfeel, elasticity mouthfeel and sweetness of the prepared steamed sponge cakes were improved, and the crumb structure was not impaired.

[0161] [Table 36]

[0162] In addition, as the results of texture analysis can be seen from Table 13, when PoAMG (JPO-172) was used in the preparation of steamed sponge rice cake, the prepared steamed sponge rice cake had reduced hardness and improved elasticity.

[0163] [Table 37]

Claims

1. 1. A method for producing a flour-based steamed product, comprising: a) providing a dough or paste comprising flour and a glucoamylase that is at least 70% identical to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; and b) A process comprising steaming the dough to produce a flour-based steamed product.

2. 2. The method of claim 1, wherein the glucoamylase is a glucoamylase variant or a mature thermostable variant of a parent glucoamylase.

3. 2. The method of claim 1, wherein the flour-based steamed product is selected from steamed buns, stuffed steamed buns (baozi), steamed twisted buns (huajuan), steamed rolls (chenzi), steamed dumplings, braised noodles (men mian), steamed sponge cakes (fa gao), steamed sponge rice cakes (mi fa gao), and Chinese New Year cakes (nian gao).

4. 2. The method of claim 1, wherein the dough or paste is made from a flour-containing material, preferably the flour is selected from the group consisting of wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour or sorghum flour, potato flour and combinations thereof.

5. 2. The method of claim 1, wherein the parent glucoamylase is obtained from a species of the genus Penicillium, preferably Penicillium oxycalum, Penicillium miczynskii, Penicillium russellii or Penicillium glaburum.

6. 2. The method of claim 1, wherein the mature variant comprises at least one amino acid modification at one or more (some) or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 65, 79, 103, 132, 327, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:

1.

7. 2. The method of claim 1, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO:1, preferably the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO:

1.

8. 2. The method of claim 1, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:1, preferably the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

9. 2. The method of claim 1, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO:1, preferably the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

10. 2. The method of claim 1, wherein the mature thermostable variant has a thermostability improvement (Td) over its parent of at least 3°C, preferably at least 4°C, 5°C, 6°C, 7°C or 8°C.

11. 2. The method of claim 1, wherein the mature thermostable variant has a relative activity at 91°C of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 compared to its parent.

12. 2. The method of claim 1, wherein the mature thermostable variant of the glucoamylase, preferably the parent glucoamylase, is at least 71% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, such as at least 72%, for example at least 73%, such as at least 74%, for example at least 75%, such as at least 76%, for example at least 77%, such as at least 78%, for example at least 79%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, such as at least 100% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

13. 13. The method according to any one of claims 1 to 12, wherein the flour-based steamed product has reduced hardness and / or improved springiness after storage at room temperature or at a low temperature of 4°C for 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6 or 7 days compared to the condition where no enzyme is added to the dough / paste but other conditions are the same.

14. 13. The method according to any one of claims 1 to 12, wherein the flour-based steamed product has improved sensory evaluation values ​​after storage at room temperature or at a low temperature of 4°C for 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6 or 7 days compared to conditions other than the addition of enzymes to the dough / paste being the same.

15. 13. The method according to any one of claims 1 to 12, wherein when the flour-based steamed product is re-steamed after storage at room temperature or at a low temperature of 4°C for 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6 or 7 days compared to when no enzyme is added to the dough / paste and all other conditions are the same, the re-steamed flour-based steamed product has an improved sensory evaluation.

16. The method according to any one of claims 1 to 12, wherein the sensory evaluation is preferably an overall evaluation, an average value of softness, springiness, whiteness of appearance, crumb structure, moisture, cohesiveness, chewiness and / or sweetness of the flour-based steamed product or the re-steamed steamed product.

17. Flour composition, dough premix or paste premix comprising flour and a thermostable glucoamylase, preferably a mature thermostable variant of a parent glucoamylase according to any of claims 1 to 12.

18. A dough comprising flour and a thermostable glucoamylase, preferably a mature thermostable variant of a parent glucoamylase according to any of claims 1 to 12.