WO2020133194A1 - 一种多菌种分步发酵制备风味性发酵豆粕的方法 - Google Patents
一种多菌种分步发酵制备风味性发酵豆粕的方法 Download PDFInfo
- Publication number
- WO2020133194A1 WO2020133194A1 PCT/CN2018/124799 CN2018124799W WO2020133194A1 WO 2020133194 A1 WO2020133194 A1 WO 2020133194A1 CN 2018124799 W CN2018124799 W CN 2018124799W WO 2020133194 A1 WO2020133194 A1 WO 2020133194A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fermentation
- soybean meal
- preparing
- fermented soybean
- saccharomyces cerevisiae
- Prior art date
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
Definitions
- the invention belongs to the technical field of soybean meal fermentation, and in particular relates to a method for preparing a flavored fermented soybean meal by step fermentation of multiple strains.
- the existing fermented soybean meal is classified into single-strain fermentation and multi-strain fermentation.
- Single-strain fermentation such as single-strain fermentation of Bacillus subtilis, single-strain fermentation of Saccharomyces cerevisiae, single-strain fermentation of lactic acid bacteria, etc.
- Multi-strain fermentation includes: Saccharomyces cerevisiae and lactic acid bacteria Bacteria fermentation, mixed fermentation of Bacillus subtilis and Saccharomyces cerevisiae and lactic acid bacteria, mixed fermentation of enzyme preparations and Saccharomyces cerevisiae and lactic acid bacteria, whether it is single strain fermentation or mixed strain fermentation, it is mostly one-step fermentation, the functionality of different strains There is no separation and good display, and there is not enough environment or action space for aerobic bacteria.
- the main purpose of the present application is to provide a method for preparing flavored fermented soybean meal by multi-strain stepwise fermentation, which aims to solve the problem that the existing soybean meal adopts one-step fermentation and the functionalities of different strains are not separated and well displayed, resulting in The problem of poor taste and flavor.
- the method for preparing a flavored fermented soybean meal by the multi-bacterial stepwise fermentation proposed in this application includes the following steps:
- Aerobic fermentation of Aspergillus oryzae Add soybean meal to water, inoculate Aspergillus oryzae with a solid dry basis ratio of 10%-100%, and control the activity of Aspergillus oryzae after inoculation to be 10 ⁇ 7cfu/g-10 ⁇ 10cfu/g, Fermentation 12-20h;
- Saccharomyces cerevisiae fermentation after the aerobic fermentation of Aspergillus oryzae, inoculate 10%-100% by weight of solid dry base of Saccharomyces cerevisiae liquid, the viability of the liquid is controlled at 10 ⁇ 7cfu/mL-10 ⁇ 10cfu/mL, Then put the material layer into the closed fermentation equipment, the surface layer is closed for oxygen isolation operation, fermentation 18h-48h, the end of the aerobic fermentation of Saccharomyces cerevisiae bacterial activity exceeds 10 ⁇ 10cfu/g;
- a method for preparing flavored fermented soybean meal by multi-strain fermentation in steps provided by the present application is inoculated with Aspergillus oryzae, saccharomyces cerevisiae liquid, and Streptococcus thermophilus liquid in sequence and fermented in sequence, through up-down cooperation between the strains Relationship, gradually increase the fermentation temperature, away from the optimal fermentation temperature of Saccharomyces cerevisiae, the yeast cells are gradually lysed, increasing the percentage of yeast wall breaking, releasing the nucleotides therein, making the fermentation and enzymolysis more thorough, reducing the molecular peptides, soybean balls The protein and ⁇ -conglycinin are degraded more thoroughly to solve the problem of sensitization of soybean antigen.
- soybean antigen The removal rate of soybean antigen is more than 90%, and other anti-nutritional factors are basically removed; the taste problem of the existing fermented soybean meal is solved.
- sensitization is positively correlated with the content of soybean antigen.
- the content of soybean antigen is mainly composed of glycinin and ⁇ -conglycinin, which is also the main factor causing sensitization.
- most of the fermented soybean meal is directly digested with industrial enzymes, resulting in a poor taste and bitter taste, and there is a high probability that bitter peptides appear on the molecular weight distribution.
- the present application improves the content ratio of compound flavor substances through different metabolites produced by step fermentation.
- the activity of the flavor protease enzyme in the fermentation end step in step 1) is not less than 100,000 IU.
- the present application ensures the aerobic fermentation of Aspergillus oryzae in the first step by controlling the enzyme activity of flavor protease in the end point of fermentation, and reduces the molecular peptides; it is easy to coordinate with the subsequent fermentation of S. cerevisiae.
- the material wet base moisture is 30%-60%, the material layer thickness is 40-100cm, the application material wet base moisture is 30%-60%, the material layer thickness is 40-100cm, It is easy to control the humidity of fermentation and maintain the fermentation temperature; there is no need to adjust the reaction temperature.
- the material needs to be turned every 20-120 minutes.
- the material is turned once every 20-120 minutes to ensure the oxygen content of the fermentation of the bottom material; and to ensure that Aspergillus oryzae has sufficient mold.
- the soybean meal in step 1) is also subjected to a pretreatment process before use, removing 43%-48% protein soybean meal, removing large particles, and then removing metal impurities that can be adsorbed by the magnet, and then crushing The particle size should be able to pass all 10 mesh screens.
- the crushed and sifted soybean meal is subjected to steam treatment again to maintain the temperature of the crushed soybean meal at 80-90°C for 10-30 minutes, and then cooled to 30-35°C.
- steam treatment is performed on the crushed soybean meal to ensure that the temperature is maintained at 80-90°C for 10-30 minutes.
- the temperature of the soybean meal is controlled, so that the soybean meal absorbs a certain amount of moisture, and heating the soybean meal facilitates subsequent fermentation.
- step 2) the materials are stacked to a height of 2-4 meters to perform the anaerobic fermentation stage of S. cerevisiae.
- the materials are stacked to a height of 2-4 meters to perform the anaerobic fermentation stage of S. cerevisiae.
- the internal materials are kept in anoxic state to ensure the progress of anaerobic fermentation.
- step 2) the thickness of the material layer is unchanged for the first 8-12 hours, and the material is turned over every hour, and then the material is kept still.
- the thickness of the material layer is unchanged in the first 8-12 hours, and the material is turned every hour, and then the material is kept still; the previous step ensures that the material layer can have sufficient nutrients; the latter step ensures the anaerobic fermentation.
- step 2) after spraying the bacterial liquid, the moisture content of the material is 40%-80%.
- step 3 the moisture content of the material after spraying the bacterial liquid is 60%.
- FIG. 1 is a microscopic view of a cell wall broken wall of a multi-strain fermentation process for preparing flavored fermented soybean meal in Example 1 of the present application.
- AMP is adenine nucleotide (adenylate)
- GMP is guanine nucleotide (guanylate)
- CMP is cytosine nucleotide (cytidine)
- UMP is uracil nucleotide (urine Glycine)
- TMP is thymine nucleotide (thymidylate)
- IMP hypoxanthine nucleotide (inosinic acid)
- a method for preparing flavored fermented soybean meal by step fermentation of multiple strains includes the following steps:
- Pretreatment process remove 43% protein soybean meal, remove large particles, and then remove metal impurities that can be adsorbed by the magnet, and then crush, the particle size after crushing should be able to pass all 10 mesh screens; crushed and sieved soybean meal After steam treatment, the temperature of the soybean meal after crushing is maintained at 80°C for 10 minutes, and then cooled to 30°C.
- Aerobic fermentation of Aspergillus oryzae Soybean meal is added to water and inoculated with Aspergillus oryzae with a solid dry basis ratio of 10%-100%, where the wet moisture of the material after inoculation is 30% and the thickness of the material layer is 40cm; and the inoculation is controlled
- the aspergillus oryzae activity is 10 ⁇ 7cfu/g after fermentation for 12h.
- the material needs to be turned every 20min; the activity of the end point of the fermentation protease is not less than 100,000IU.
- Saccharomyces cerevisiae fermentation after the aerobic fermentation of Aspergillus oryzae, inoculate 10% by weight of solid dry base of Saccharomyces cerevisiae bacterial liquid, the bacterial liquid vigor is controlled at 10 ⁇ 7cfu/mL, after spraying the bacterial liquid, the material is wet based Water content 40%; then put the material layer in the closed fermentation equipment, stack the material to a height of 2 meters for the anaerobic fermentation stage of Saccharomyces cerevisiae (surface layer is closed for oxygen isolation operation), fermentation 18h, the thickness of the material layer is unchanged for the first 8h , Turn over the material once every hour, and then keep the material still; the end-strain of aerobic fermentation of Saccharomyces cerevisiae exceeds 10 ⁇ 10cfu/g;
- thermophilic S. thermophilus bacterial liquid in which the moisture content of the material after spraying the bacterial liquid is 60%, and the vigor of the bacterial liquid is controlled at 10 ⁇ 7 cfu/mL, the fermentation temperature rises gradually, fermentation 18h, until the fermentation temperature reaches 50 °C, and maintain this temperature until the pH is below 4.5 is the end point.
- the percentage of broken yeast wall of the flavored fermented soybean meal prepared in this Example 1 is 60%;
- Table 1 below is the data integration table of different molecular weight substances in the molecular weight distribution diagram of the flavored fermented soybean meal and the soybean meal in the market of Example 1;
- Volatile flavor substances content Ethyl acetate 1.2% Acetoin 0.16% Maltol 1.6% Ethyl palmitate 0.5% Benzyl alcohol 1.1% 1-octene-3-ol 3.2%
- the percentage content is the percentage of total volatile substances measured by GC-MS.
- a method for preparing flavored fermented soybean meal by step fermentation of multiple strains includes the following steps:
- Pretreatment process remove 48% protein soybean meal, remove large particles, and then remove metal impurities that can be adsorbed by the magnet, and then pulverize.
- the particle size after pulverization should be able to pass through a 10-mesh sieve; After steam treatment, the temperature of the soybean meal after crushing is maintained at 90°C for 30 minutes, and then cooled to 35°C.
- Saccharomyces cerevisiae fermentation After the aerobic fermentation of Aspergillus oryzae, inoculate 100% by weight of solid dry base of Saccharomyces cerevisiae liquid, the viability of the liquid liquid is controlled at 10 ⁇ 10cfu/mL, after spraying the liquid, the material is wet based The water content is 80%; then put the material layer in the closed fermentation equipment, stack the material to a height of 4 meters for the anaerobic fermentation stage of Saccharomyces cerevisiae (the surface layer is closed for oxygen isolation operation), fermentation 48h, the thickness of the material layer is unchanged for the first 12h , Turn over the material once every hour, and then keep the material still; the end-strain of aerobic fermentation of Saccharomyces cerevisiae exceeds 10 ⁇ 10cfu/g;
- Lactobacillus fermentation After the fermentation of Saccharomyces cerevisiae, inoculate 100% by weight of the solid dry base of thermophilic Streptococcus liquid, in which the moisture content of the material after spraying the liquid is 60%, and the viability of the liquid is controlled at 10 ⁇ 9cfu /mL, the fermentation temperature gradually rises, fermentation for 80h, until the fermentation temperature reaches 50 °C, and maintain this temperature until the pH is below 4.5 is the end point.
- the percentage of broken walls of the flavored fermented soybean meal yeast prepared in Example 2 was 66%.
- Volatile flavor substances content Ethyl acetate 1.1% Acetoin 0.17% Maltol 1.8% Ethyl palmitate 0.8% Benzyl alcohol 1.2% 1-octene-3-ol 3.6%
- a method for preparing flavored fermented soybean meal by step fermentation of multiple strains includes the following steps:
- Pre-treatment process 45% protein soybean meal, remove large particles, and then remove metal impurities that can be adsorbed by the magnet, and then crush, the particle size after crushing should be able to pass all 10 mesh screen; After steam treatment, the temperature of the soybean meal after crushing is maintained at 85°C for 20 minutes, and then cooled to 32.5°C.
- Aerobic fermentation of Aspergillus oryzae Add soybean meal to water and inoculate Aspergillus oryzae with a solid dry basis ratio of 10%-100%. Among them, the wet moisture of the material after inoculation is 45% and the thickness of the material layer is 70cm; and control the inoculation After the Aspergillus oryzae activity is 10 ⁇ 8cfu/g, fermentation for 16h, in the fermentation process, the material needs to be turned every 70min; the end of the fermentation material flavor protease enzyme activity is not less than 100,000IU.
- Saccharomyces cerevisiae fermentation After the aerobic fermentation of Aspergillus oryzae, inoculate 55% by weight of the solid dry base of S. cerevisiae liquid, and the vigor of the liquid is controlled at 10 ⁇ 9cfu/mL.
- the material After spraying the liquid, the material is wet based The water content is 60%; then put the material layer in the closed fermentation equipment, stack the material to a height of 3 meters for the anaerobic fermentation stage of Saccharomyces cerevisiae (the surface layer is closed for oxygen isolation operation), fermentation 33h, the thickness of the material layer is unchanged for the first 10h , Turn over the material once every hour, and then keep the material still; the end-strain of aerobic fermentation of Saccharomyces cerevisiae exceeds 10 ⁇ 10cfu/g;
- the breakage ratio of the flavored fermented soybean meal yeast prepared in Example 3 was 69%.
- Volatile flavor substances content Ethyl acetate 1.3% Acetoin 0.18% Maltol 1.6% Ethyl palmitate 0.7% Benzyl alcohol 1.5% 1-octene-3-ol 3.8%
- a method for preparing flavored fermented soybean meal by step fermentation of multiple strains includes the following steps:
- Pretreatment process remove 42% protein soybean meal, remove large particles, and then remove metal impurities that can be adsorbed by the magnet, and then pulverize, the particle size after pulverization should be able to pass all 10 mesh screens; After steam treatment, the temperature of the soybean meal after crushing was maintained at 79°C for 9 minutes, and then cooled to 29°C.
- Aerobic fermentation of Aspergillus oryzae Soybean meal is added to water, inoculated with 9% solid dry basis of Aspergillus oryzae, wherein the wet moisture of the material after inoculation is 30%-60%, and the thickness of the material layer is 39cm; and the inoculation is controlled
- the aspergillus oryzae activity is 9 ⁇ 7cfu/g, fermentation is 11h, during the fermentation process, the material needs to be turned every 19min; the activity of the end point of the fermentation protease enzyme is not less than 100,000IU.
- Saccharomyces cerevisiae fermentation After the aerobic fermentation of Aspergillus oryzae, inoculate 9% by weight of solid dry base of Saccharomyces cerevisiae liquid, the viability of the liquid liquid is controlled at 9 ⁇ 7cfu/mL, after spraying the liquid, the material is wet based Moisture content 39%; then put the material layer in the closed fermentation equipment, pile up the material to a height of 1 meter for the anaerobic fermentation stage of Saccharomyces cerevisiae (the surface layer is closed for oxygen isolation operation), fermentation 17h, the thickness of the material layer is unchanged for the first 7h , Turn over the material once every hour, and then keep the material still; the end-point bacterial activity of S. cerevisiae aerobic fermentation is 9 ⁇ 10cfu/g;
- the ratio of the broken wall of the flavored fermented soybean meal yeast prepared in this Comparative Example 1 was 44%.
- Volatile flavor substances content Ethyl acetate 0.4% Acetoin 0.05% Maltol 0.9% Ethyl palmitate 0.3% Benzyl alcohol 0.4% 1-octene-3-ol 1.3%
- a method for preparing flavored fermented soybean meal by step fermentation of multiple strains includes the following steps:
- Pre-treatment process remove 49% protein soybean meal, remove large particles, and then remove metal impurities that can be adsorbed by the magnet, and then crush, the crushed particle size should be able to pass all 10 mesh screen; crushed and sieved soybean meal After steam treatment, the temperature of the soybean meal after crushing is maintained at 91°C for 31 minutes, and then cooled to 36°C.
- Aerobic fermentation of Aspergillus oryzae Soybean meal is added to water, inoculated with 100% solid dry basis of Aspergillus oryzae, wherein the wet moisture of the material after inoculation is 29% and the thickness of the material layer is 39cm; and the rice after inoculation is controlled Aspergillus activity is 11 ⁇ 10cfu/g, fermentation for 21h, during the fermentation process, the material needs to be turned every 121min; the end point of the fermentation protease enzyme activity is not less than 100,000IU.
- Saccharomyces cerevisiae fermentation After the aerobic fermentation of Aspergillus oryzae, inoculate 100% by weight of solid dry base of Saccharomyces cerevisiae liquid, the viability of the liquid is controlled at 11 ⁇ 10cfu/mL, after spraying the liquid, the material is wet based Moisture content 81%; Then put the material layer in the closed fermentation equipment, stack the material to a height of 2-4 meters for the anaerobic fermentation stage of Saccharomyces cerevisiae (the surface layer is closed for oxygen isolation operation), fermentation 49h, thickness of the first 13h layer No change, turn over the material once every hour, and then keep the material still; the activity of the aerobic fermentation end point of Saccharomyces cerevisiae exceeds 10 ⁇ 10cfu/g;
- Lactobacillus fermentation After the fermentation of Saccharomyces cerevisiae, inoculate 100% by weight of the solid dry base of thermophilic Streptococcus liquid, in which the moisture content of the material after spraying the liquid is 60%, and the viability of the liquid is controlled at 11 ⁇ 9cfu /mL, the fermentation temperature rises gradually, fermentation 81h, until the fermentation temperature reaches 50 °C, and maintain this temperature until the pH is below 4.5 is the end point.
- the ratio of the broken wall of the flavor fermented soybean meal yeast prepared in this Comparative Example 2 is 45%.
- the following table 6 shows the content of the volatile flavor substances in the comparative example 1 in the total volatile substances:
- Volatile flavor substances content Ethyl acetate 0.5% Acetoin 0.06% Maltol 0.8% Ethyl palmitate 0.3% Benzyl alcohol 0.3% 1-octene-3-ol 1.6%
- Example 1 the nucleotide content of Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2 are shown in Table 7 below:
- Example 1 , Example 2 and Example 3 flavored fermented soybean meal solves the problem of sensitization of soybean antigen, the soybean antigen removal rate reaches more than 90%, and other anti-nutritional factors are basically removed; the taste problem of the existing fermented soybean meal is solved.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Beans For Foods Or Fodder (AREA)
Abstract
多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：1)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为10％-100％的米曲霉霉菌，发酵12-20h；2)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例10％-100％重量的洒酿酒酵母菌液，然后将料层放入密闭发酵设备中，表层密闭进行隔氧操作，发酵18h-48h，酿酒酵母好氧发酵终点菌活超过10^10cfu/g；3)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例10％-100％重量的嗜热链球菌菌液，发酵18-80h，直至发酵温度达到50℃。该方法解决了现有的豆粕发酵中不同菌种的功能性没有分开及良好的展现、导致口感和风味性较差的问题。
Description
本发明属于豆粕发酵技术领域，具体地说，涉及一种多菌种分步发酵制备风味性发酵豆粕的方法。
现有的发酵豆粕分单菌种发酵及多菌种发酵，单菌种发酵如枯草芽孢单菌株发酵，酿酒酵母单菌株发酵，乳酸菌单菌株发酵等，多菌种发酵有：酿酒酵母加乳酸菌混菌发酵，枯草芽孢杆菌加酿酒酵母菌加乳酸菌混合发酵，酶制剂加酿酒酵母加乳酸菌混合发酵等，无论是单菌种发酵还是混合菌种发酵，多为一步式发酵，不同菌种的功能性没有分开及良好的展现，没有给与好氧菌充分的环境或作用空间。现有常规发酵豆粕多使用工业性酶制剂，容易暴露疏水性氨基酸末端，造成大豆蛋白呈现苦味，而在一步式发酵过程中由于发酵及酶解的不彻底，中分子肽较多，大豆球蛋白和β-伴大豆球蛋白降解不彻底，使得口感和风味性较差。
发明内容
本申请的主要目的是提供一种多菌种分步发酵制备风味性发酵豆粕的方法，旨在解决现有的豆粕采用一步式发酵中，不同菌种的功能性没有分开及良好的展现，导致口感和风味性较差的问题。
为实现上述目的，本申请提出的多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：
1)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为10％-100％的米曲霉霉菌，控制接种后的米曲霉菌活为10^7cfu/g-10^10cfu/g，发酵12-20h；
2)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例10％-100％重量的洒酿酒酵母菌液，菌液活力控制在10^7cfu/mL-10^10cfu/mL，然后将料层放入密闭发酵设备中，表层密闭进行隔氧操作，发酵18h-48h，酿酒酵母好氧发酵终点菌活超过10^10cfu/g；
3)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例10％-100％重量的嗜热链球菌菌液，菌液活力控制在10^7cfu/mL-10^9cfu/mL，发酵温度逐步上升，发酵18-80h，直至发酵温度达到50℃，并维持此温度至pH低于4.5即为终点。本申请所提供的一种多菌种分步发酵制备风味性发酵豆粕的方法，依次接种米曲霉霉菌、洒酿酒酵母菌液、嗜热链球菌菌液并依次发酵，通过菌种间的上下协作关系，逐步提高发酵温度，脱离酿酒酵母最适发酵温度，酵母细胞逐步裂解，提高了酵母破壁比例，释放其中的核苷酸，使得发酵及酶解更彻底，减少了中分子肽，大豆球蛋白和β-伴大豆球蛋白降解更彻底，解决大豆抗原的致敏性问题，将大豆抗原去除率达到90％以上，其他抗营养因子基本全部去除；解决了现有发酵豆粕的口感问题。这里致敏性和大豆抗原的含量呈正相关，大豆抗原含量主要由大豆球蛋白和β-伴大豆球蛋白组成，也是造成致敏性的主要因素。而且目前的发酵豆粕多使用工业酶直接酶切，造成发酵豆粕口感较差，多有苦味，分子量分布上面呈现出苦味肽比较大概率出现。同时，本申请通过分步发酵产生的不同代谢产物，提升复合风味物质的含量比例。
可选地，步骤1)中发酵终点物料风味蛋白酶酶活不低于100000IU。本申请通过控制发酵终点物料风味蛋白酶酶活，保证第一步米曲霉好氧发酵充分，减少了中分子肽；便于与后续的酿酒酵母发酵协调。
可选地，步骤1)中接种后物料湿基水分为30％-60％，料层厚度为40-100cm本申请通过物料湿基水分为30％-60％，料层厚度为40-100cm，便于控制发酵的湿度和保持发酵温度；不需要再进行调节反应温度。
可选地，步骤1)中的发酵过程中，需要每20-120min翻料一次。本申请通过20-120min翻料一次，保证了底层物料发酵的氧含量；保证米曲霉霉菌充分。
可选地，步骤1)中的豆粕，在使用前还进行前处理工序，将43％-48％蛋白豆粕，去除大颗粒，再经过去除可被磁铁吸附的金属杂质，然后粉碎，粉碎后的粒度应能全部通过10目筛网。
可选地，粉碎过筛后的豆粕再经过蒸汽处理，使粉碎后豆粕温度保持在80-90℃，并持续10-30分钟，然后冷却至30-35℃。本申请通过对粉碎后的豆粕进行蒸汽处理，保证温度保持在80-90℃，并持续10-30分钟，控制了豆 粕的温度，使得豆粕吸附一定的水分，同时加热豆粕便于后续的发酵充分。
可选地，步骤2)中，将物料堆积至2-4米高度后进行酿酒酵母的厌氧发酵阶段。本申请通过将物料堆积至2-4米，使得内部物料保持在缺氧状态，保证了厌氧发酵的进行。
可选地，步骤2)中，前8-12h料层厚度不变，每小时翻料一次，之后保持物料静止。本申请通过在前8-12h料层厚度不变，每小时翻料一次，之后保持物料静止；前一个步骤保证料层能有具有充分的养分；后一个步骤保证厌氧发酵的进行。
可选地，步骤2)中，喷洒菌液后，物料湿基含水量40％-80％。
可选地，步骤3)中，喷洒菌液后物料湿基含水量60％。
有益效果：本申请技术方案中，通过分布发酵工序，依次接种米曲霉霉菌、洒酿酒酵母菌液、嗜热链球菌菌液并依次发酵，通过菌种间的上下协作关系，使得发酵及酶解更彻底，减少了中分子肽较多，大豆球蛋白和β-伴大豆球蛋白降解更彻底，解决了大豆抗原的致敏性问题，将大豆抗原去除率达到90％以上，其他抗营养因子基本全部去除；使得口感和风味性更佳。
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图示出的结构获得其他的附图。
图1为本申请实施例1中多菌种分步发酵制备风味性发酵豆粕的细胞壁破壁显微镜视图。
本申请目的的实现、功能特点及优点将结合实施例，参照附图做进一步说明。
下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请的一部分实施例， 而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
需要说明，本申请中所用原料、设备，若无特别说明，均为本领域的常用原料、设备，均来自市售产品。本申请中所用方法，若无特别说明，均为本领域技术人员所知晓的常规方法。
其中，AMP为腺嘌呤核苷酸(腺苷酸)、GMP为鸟嘌呤核苷酸(鸟苷酸)、CMP为胞嘧啶核苷酸(胞苷酸)、UMP为尿嘧啶核苷酸(尿苷酸)、TMP为胸腺嘧啶核苷酸(胸苷酸)及IMP为次黄嘌呤核苷酸(肌苷酸，)
另外，本申请各个实施例之间的技术方案可以相互结合，但是必须是以本领域普通技术人员能够实现为基础，当技术方案的结合出现相互矛盾或无法实现时应当认为这种技术方案的结合不存在，也不在本申请要求的保护范围之内。
实施例1
一种多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：
1)前处理工序：将43％蛋白豆粕，去除大颗粒，再经过去除可被磁铁吸附的金属杂质，然后粉碎，粉碎后的粒度应能全部通过10目筛网；粉碎过筛后的豆粕再经过蒸汽处理，使粉碎后豆粕温度保持在80℃，并持续10分钟，然后冷却至30℃。
2)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为10％-100％的米曲霉霉菌，其中，接种后物料湿基水分为30％，料层厚度为40cm；并控制接种后的米曲霉菌活为10^7cfu/g，发酵12h，在发酵过程中，需要每20min翻料一次；发酵终点物料风味蛋白酶酶活不低于100000IU。
3)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例10％重量的洒酿酒酵母菌液，菌液活力控制在10^7cfu/mL，其中喷洒菌液后，物料湿基含水量40％；然后将料层放入密闭发酵设备中，将物料堆积至2米高度进行酿酒酵母的厌氧发酵阶段(表层密闭进行隔氧操作)，发酵18h，前8h料层厚度不变，每小时翻料一次，之后保持物料静止；酿酒酵母好氧发酵终点菌活超过10^10cfu/g；
4)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例10％重量的嗜热链球菌菌液，其中，喷洒菌液后物料湿基含水量60％，菌液活力控制在10^7 cfu/mL，发酵温度逐步上升，发酵18h，直至发酵温度达到50℃，并维持此温度至pH低于4.5即为终点。
参见图1，本实施例1制得的风味性发酵豆粕酵母破壁比例为60％；下表1为实施例1风味性发酵豆粕以及市面上的豆粕分子量分布图中不同分子量物质数据积分表；
下表2为实施例1的挥发性风味物质占总挥发性物质的含量：
| 挥发性风味物质 | 含量 |
| 乙酸乙酯 | 1.2％ |
| 乙偶姻 | 0.16％ |
| 麦芽酚 | 1.6％ |
| 棕榈酸乙酯 | 0.5％ |
| 苯甲醇 | 1.1％ |
| 1-辛烯-3-醇 | 3.2％ |
注:百分含量为用GC-MS测量出来的挥发性风味物质占总挥发性物质的百分比。
实施例2
一种多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：
1)前处理工序：将48％蛋白豆粕，去除大颗粒，再经过去除可被磁铁吸附的金属杂质，然后粉碎，粉碎后的粒度应能全部通过10目筛网；粉碎过筛后的豆粕再经过蒸汽处理，使粉碎后豆粕温度保持在90℃，并持续30分钟，然后冷却至35℃。
2)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为100％的米曲霉霉菌，其中，接种后物料湿基水分为60％，料层厚度为100cm；并控制接种后的米曲霉菌活为10^10cfu/g，发酵12-20h，在发酵过程中，需要每120min翻料一次；发酵终点物料风味蛋白酶酶活不低于100000IU。
3)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例100％重 量的洒酿酒酵母菌液，菌液活力控制在10^10cfu/mL，其中喷洒菌液后，物料湿基含水量80％；然后将料层放入密闭发酵设备中，将物料堆积至4米高度进行酿酒酵母的厌氧发酵阶段(表层密闭进行隔氧操作)，发酵48h，前12h料层厚度不变，每小时翻料一次，之后保持物料静止；酿酒酵母好氧发酵终点菌活超过10^10cfu/g；
4)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例100％重量的嗜热链球菌菌液，其中，喷洒菌液后物料湿基含水量60％，菌液活力控制在10^9cfu/mL，发酵温度逐步上升，发酵80h，直至发酵温度达到50℃，并维持此温度至pH低于4.5即为终点。
本实施例2制得的风味性发酵豆粕酵母破壁比例为66％。
下表3为实施例2的挥发性风味物质占总挥发性物质的含量：
| 挥发性风味物质 | 含量 |
| 乙酸乙酯 | 1.1％ |
| 乙偶姻 | 0.17％ |
| 麦芽酚 | 1.8％ |
| 棕榈酸乙酯 | 0.8％ |
| 苯甲醇 | 1.2％ |
| 1-辛烯-3-醇 | 3.6％ |
实施例3
一种多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：
1)前处理工序：将45％蛋白豆粕，去除大颗粒，再经过去除可被磁铁吸附的金属杂质，然后粉碎，粉碎后的粒度应能全部通过10目筛网；粉碎过筛后的豆粕再经过蒸汽处理，使粉碎后豆粕温度保持在85℃，并持续20分钟，然后冷却至32.5℃。
2)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为10％-100％的米曲霉霉菌，其中，接种后物料湿基水分为45％，料层厚度为70cm；并控制接种后的米曲霉菌活为10^8cfu/g，发酵16h，在发酵过程中，需要每70min翻料一次；发酵终点物料风味蛋白酶酶活不低于100000IU。
3)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例55％重 量的洒酿酒酵母菌液，菌液活力控制在10^9cfu/mL，其中喷洒菌液后，物料湿基含水量60％；然后将料层放入密闭发酵设备中，将物料堆积至3米高度进行酿酒酵母的厌氧发酵阶段(表层密闭进行隔氧操作)，发酵33h，前10h料层厚度不变，每小时翻料一次，之后保持物料静止；酿酒酵母好氧发酵终点菌活超过10^10cfu/g；
4)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例55％重量的嗜热链球菌菌液，其中，喷洒菌液后物料湿基含水量60％，菌液活力控制在10^8cfu/mL，发酵温度逐步上升，发酵49h，直至发酵温度达到50℃，并维持此温度至pH低于4.5即为终点。
本实施例3制得的风味性发酵豆粕酵母破壁比例为69％。
下表4为实施例3的挥发性风味物质占总挥发性物质的含量：
| 挥发性风味物质 | 含量 |
| 乙酸乙酯 | 1.3％ |
| 乙偶姻 | 0.18％ |
| 麦芽酚 | 1.6％ |
| 棕榈酸乙酯 | 0.7％ |
| 苯甲醇 | 1.5％ |
| 1-辛烯-3-醇 | 3.8％ |
对比例1
一种多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：
1)前处理工序：将42％蛋白豆粕，去除大颗粒，再经过去除可被磁铁吸附的金属杂质，然后粉碎，粉碎后的粒度应能全部通过10目筛网；粉碎过筛后的豆粕再经过蒸汽处理，使粉碎后豆粕温度保持在79℃，并持续9分钟，然后冷却至29℃。
2)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为9％的米曲霉霉菌，其中，接种后物料湿基水分为30％-60％，料层厚度为39cm；并控制接种后的米曲霉菌活为9^7cfu/g，发酵11h，在发酵过程中，需要每19min翻料一次；发酵终点物料风味蛋白酶酶活不低于100000IU。
3)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例9％重量 的洒酿酒酵母菌液，菌液活力控制在9^7cfu/mL，其中喷洒菌液后，物料湿基含水量39％；然后将料层放入密闭发酵设备中，将物料堆积至1米高度进行酿酒酵母的厌氧发酵阶段(表层密闭进行隔氧操作)，发酵17h，前7h料层厚度不变，每小时翻料一次，之后保持物料静止；酿酒酵母好氧发酵终点菌活为9^10cfu/g；
4)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例9％重量的嗜热链球菌菌液，其中，喷洒菌液后物料湿基含水量60％，菌液活力控制在10^6cfu/mL，发酵温度逐步上升，发酵17h，直至发酵温度达到50℃，并维持此温度至pH低于4.5即为终点。
本对比例1制得的风味性发酵豆粕酵母破壁比例为44％。
下表5为对比例1的挥发性风味物质占总挥发性物质的含量：
| 挥发性风味物质 | 含量 |
| 乙酸乙酯 | 0.4％ |
| 乙偶姻 | 0.05％ |
| 麦芽酚 | 0.9％ |
| 棕榈酸乙酯 | 0.3％ |
| 苯甲醇 | 0.4％ |
| 1-辛烯-3-醇 | 1.3％ |
对比例2
一种多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：
1)前处理工序：将49％蛋白豆粕，去除大颗粒，再经过去除可被磁铁吸附的金属杂质，然后粉碎，粉碎后的粒度应能全部通过10目筛网；粉碎过筛后的豆粕再经过蒸汽处理，使粉碎后豆粕温度保持在91℃，并持续31分钟，然后冷却至36℃。
2)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为100％的米曲霉霉菌，其中，接种后物料湿基水分为29％，料层厚度为39cm；并控制接种后的米曲霉菌活为11^10cfu/g，发酵21h，在发酵过程中，需要每121min翻料一次；发酵终点物料风味蛋白酶酶活不低于100000IU。
3)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例100％重 量的洒酿酒酵母菌液，菌液活力控制在11^10cfu/mL，其中喷洒菌液后，物料湿基含水量81％；然后将料层放入密闭发酵设备中，将物料堆积至2-4米高度进行酿酒酵母的厌氧发酵阶段(表层密闭进行隔氧操作)，发酵49h，前13h料层厚度不变，每小时翻料一次，之后保持物料静止；酿酒酵母好氧发酵终点菌活超过10^10cfu/g；
4)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例100％重量的嗜热链球菌菌液，其中，喷洒菌液后物料湿基含水量60％，菌液活力控制在11^9cfu/mL，发酵温度逐步上升，发酵81h，直至发酵温度达到50℃，并维持此温度至pH低于4.5即为终点。
本对比例2制得的风味性发酵豆粕酵母破壁比例为45％。
下表6为对比例1的挥发性风味物质占总挥发性物质的含量：
| 挥发性风味物质 | 含量 |
| 乙酸乙酯 | 0.5％ |
| 乙偶姻 | 0.06％ |
| 麦芽酚 | 0.8％ |
| 棕榈酸乙酯 | 0.3％ |
| 苯甲醇 | 0.3％ |
| 1-辛烯-3-醇 | 1.6％ |
其中，实施例1、实施例2、实施例3和对比例1、对比例2核苷酸含量如下表7所示：
本实施例1、实施例2、实施例3、对比例1、对比例2和市面上其他产品的大豆抗原成分如下表8所示：
通过上面数据可以得知，实施例1、实施例2和实施例3风味性发酵豆粕酵母破壁比例大大高于对比例1(采用工艺参数低于本申请的工艺范围)和对比例2(采用工艺参数高于本申请的工艺范围)的破壁比例。实施例1、实施例2和实施例3风味性发酵豆粕的核苷酸含量大大高于对比例1、对比例2以及市面上的的核苷酸含量；表明实施例1、实施例2和实施例3风味性发酵豆粕破壁比例更高，释放的核苷酸更多，挥发性风味物质占总挥发性物质的含量更高；在发酵中发酵、分解的更彻底。同时，实施例1、实施例2和实施例3风味性发酵豆粕的大豆抗原含量大大小于对比例1、对比例2以及市面上的 大豆粉、豆粕和大豆浓缩蛋白的大豆抗原含量；实施例1、实施例2和实施例3风味性发酵豆粕解决了大豆抗原的致敏性问题，将大豆抗原去除率达到90％以上，其他抗营养因子基本全部去除；解决了现有发酵豆粕的口感问题。
以上所述仅为本申请的优选实施例，并非因此限制本申请的专利范围，凡是在本申请的构思下，利用本申请说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本申请的专利保护范围内。
Claims (10)
- 一种多菌种分步发酵制备风味性发酵豆粕的方法，包括以下步骤：1)米曲霉好氧发酵：将豆粕加入水中，接种固态干基比例为10％-100％的米曲霉霉菌，控制接种后的米曲霉菌活10^7cfu/g-10^10cfu/g，发酵12-20h；2)酿酒酵母发酵：米曲霉好氧发酵结束后，接种占固态干基比例10％-100％重量的洒酿酒酵母菌液，菌液活力控制在10^7cfu/mL-10^10cfu/mL，然后将料层放入密闭发酵设备中，表层密闭进行隔氧操作，发酵18h-48h，酿酒酵母好氧发酵终点菌活超过10^10cfu/g；3)乳酸菌发酵：酿酒酵母发酵结束后，接种占固态干基比例10％-100％重量的嗜热链球菌菌液，菌液活力控制在10^7cfu/mL-10^9cfu/mL，发酵温度逐步上升，发酵18-80h，直至发酵温度达到50℃，并维持此温度至pH低于4.5即为终点。
- 根据权利要求1所述的多菌种分步发酵制备风味性发酵豆粕的方法，其中，步骤1)中发酵终点物料风味蛋白酶酶活不低于100000IU。
- 根据权利要求1所述的多菌种分步发酵制备风味性发酵豆粕的方法，其中，步骤1)中接种后物料湿基水分为30％-60％，料层厚度为40-100cm。
- 根据权利要求1所述的多菌种分步发酵制备风味性发酵豆粕的方法，其中，步骤1)中的发酵过程中，需要每20-120min翻料一次。
- 根据权利要求1或2或3或4所述的多菌种分步发酵制备风味性发酵豆粕的方法，步骤1)中的豆粕，在使用前还进行前处理工序，将43％-48％蛋白豆粕，去除大颗粒，再经过去除的金属杂质，然后粉碎，粉碎后的粒度应能全部通过10目筛网。
- 根据权利要求5所述的多菌种分步发酵制备风味性发酵豆粕的方法，前处理工序中，粉碎过筛后的豆粕再经过蒸汽处理，使粉碎后豆粕温度保持在80-90℃，并持续10-30分钟，然后冷却至30-35℃。
- 根据权利要求1或2或3或4所述的多菌种分步发酵制备风味性发酵豆粕的方法，步骤2)中，将物料堆积至2-4米高度后进行酿酒酵母的厌氧发酵阶段。
- 根据权利要求7所述的多菌种分步发酵制备风味性发酵豆粕的方法， 其中，步骤2)中，前8-12h料层厚度不变，每小时翻料一次，之后保持物料静止。
- 根据权利要求1或2或3或4所述的多菌种分步发酵制备风味性发酵豆粕的方法，步骤2)中，喷洒菌液后，物料湿基含水量40％-80％。
- 根据权利要求1或2或3或4所述的多菌种分步发酵制备风味性发酵豆粕的方法，步骤3)中，喷洒菌液后物料湿基含水量60％。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2018/124799 WO2020133194A1 (zh) | 2018-12-28 | 2018-12-28 | 一种多菌种分步发酵制备风味性发酵豆粕的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2018/124799 WO2020133194A1 (zh) | 2018-12-28 | 2018-12-28 | 一种多菌种分步发酵制备风味性发酵豆粕的方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2020133194A1 true WO2020133194A1 (zh) | 2020-07-02 |
Family
ID=71129501
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2018/124799 WO2020133194A1 (zh) | 2018-12-28 | 2018-12-28 | 一种多菌种分步发酵制备风味性发酵豆粕的方法 |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2020133194A1 (zh) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103478417A (zh) * | 2013-08-16 | 2014-01-01 | 宁波中瑞生物科技有限公司 | 一种二次接种分段固态发酵生产发酵豆粕的方法 |
| CN104000006A (zh) * | 2014-04-28 | 2014-08-27 | 福建师范大学 | 一种三阶段混菌固态发酵制备酒糟蛋白饲料的方法 |
| CN104012755A (zh) * | 2014-06-12 | 2014-09-03 | 江南大学 | 一种两步发酵高效生产发酵豆粕的方法 |
| CN104186935A (zh) * | 2014-08-01 | 2014-12-10 | 威海金牌生物科技股份有限公司 | 复合菌液发酵豆粕的方法 |
| CN107343561A (zh) * | 2017-06-27 | 2017-11-14 | 山东中阳生物科技有限公司 | 一种分步发酵制备生物活性饲料的方法 |
| CN108056272A (zh) * | 2018-01-03 | 2018-05-22 | 福建傲农生物科技集团股份有限公司 | 一种新型发酵豆粕及其制备工艺与应用 |
-
2018
- 2018-12-28 WO PCT/CN2018/124799 patent/WO2020133194A1/zh active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103478417A (zh) * | 2013-08-16 | 2014-01-01 | 宁波中瑞生物科技有限公司 | 一种二次接种分段固态发酵生产发酵豆粕的方法 |
| CN104000006A (zh) * | 2014-04-28 | 2014-08-27 | 福建师范大学 | 一种三阶段混菌固态发酵制备酒糟蛋白饲料的方法 |
| CN104012755A (zh) * | 2014-06-12 | 2014-09-03 | 江南大学 | 一种两步发酵高效生产发酵豆粕的方法 |
| CN104186935A (zh) * | 2014-08-01 | 2014-12-10 | 威海金牌生物科技股份有限公司 | 复合菌液发酵豆粕的方法 |
| CN107343561A (zh) * | 2017-06-27 | 2017-11-14 | 山东中阳生物科技有限公司 | 一种分步发酵制备生物活性饲料的方法 |
| CN108056272A (zh) * | 2018-01-03 | 2018-05-22 | 福建傲农生物科技集团股份有限公司 | 一种新型发酵豆粕及其制备工艺与应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107937292B (zh) | 一种酿酒酵母培养物及其发酵工艺 | |
| CN106173193A (zh) | 一种固态发酵豆粕的方法 | |
| CN102633865A (zh) | 一种恩拉霉素发酵液的预处理方法及以其制备恩拉霉素预混剂的方法 | |
| CN110591943B (zh) | 一株产复合酶枯草芽孢杆菌,组合物及其应用 | |
| TWI706036B (zh) | 穀物粉中濃縮蛋白質的方法 | |
| CN103478417A (zh) | 一种二次接种分段固态发酵生产发酵豆粕的方法 | |
| CN106754474A (zh) | 一种抗生素菌渣厌氧发酵促进剂及其制备与应用 | |
| CN107535671A (zh) | 提高瘤胃蛋白利用率的微生物发酵黄酒糟饲料及制备方法 | |
| CN111334407A (zh) | 一种深度发酵酵母制备酵母培养物的工艺技术及工艺设备 | |
| CN104232552B (zh) | 一种清洁生产谷氨酸钠的环保工艺 | |
| CN110384178B (zh) | 基于酒糟制备的乳酸菌培养物及其在动物饲料中的应用 | |
| CN100569946C (zh) | 热带假丝酵母菌株的分离及其用于木糖醇生产的方法 | |
| CN106173189A (zh) | 一种固态发酵棉粕的方法 | |
| WO2020133194A1 (zh) | 一种多菌种分步发酵制备风味性发酵豆粕的方法 | |
| CN105713851A (zh) | 一株拜氏梭菌及其应用 | |
| CN115005327B (zh) | 一种发酵型饲用载体及其制备方法 | |
| CN102168024A (zh) | 一种金龟子绿僵菌及其应用 | |
| CN112175857B (zh) | 植物乳酸菌固体菌剂及其制备方法和应用 | |
| CN1745647A (zh) | 一种啤酒糟生产富酶蛋白饲料的方法 | |
| CN103952334B (zh) | 一种微生物发酵生产l-赤藓酮糖的菌株hd385和方法 | |
| CN105778919A (zh) | 巨大芽孢杆菌-壳聚糖微胶囊及其制备方法 | |
| CN105462837A (zh) | 一种复合有机氮源及其制备方法 | |
| CN109744367A (zh) | 资源化利用小麦酒糟生产饲料原料的方法及饲料原料 | |
| CN108782948A (zh) | 一种酵母源功能性蛋白及其制备方法 | |
| CN112159762B (zh) | 一种尖孢镰刀菌swh-3及其发酵生产黄姜皂素的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 18945216 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 18945216 Country of ref document: EP Kind code of ref document: A1 |