WO2012022112A1

WO2012022112A1 - Sufu ripening agent and method for quickly preparing sufu by using the same

Info

Publication number: WO2012022112A1
Application number: PCT/CN2011/001235
Authority: WO
Inventors: Jianming Wang; Shaohua Zhao
Original assignee: Dsm Ip Assets B.V.
Priority date: 2010-08-20
Filing date: 2011-07-27
Publication date: 2012-02-23
Also published as: CN102370112A

Abstract

The present invention relates to a method of preparing preserved bean curd characterizing in that at least one protease is added into soybean milk before the mould is inoculated. It further relates to the use of protease in a method of preparing preserved bean curd. The method overcomes the shortcoming of existing technology to provide a quicker ripening production process compared to the traditional method of preserved bean curd.

Description

SUFU RIPENING AGENT AND METHOD FOR QUICKLY PREPARING SUFU

BY USING THE SAME

Field of the invention

5 This invention relates to the field of food production. In particular, it relates to a novel

method for producing preserved bean curd (also called as sufu as well as other names). More particularly, it is a method of taking soybean and products thereof as raw materials to produce preserved bean curd in a shorter time by using a ripening-promoting agent for sufu.

10

Background of the invention

Preserved bean curd, also called as Sufu, fermented soybean curd, and so on, is one of soybean products, which is prepared by changing the flavor of vegetable proteins with

15 microbial fermentation method. Preserved bean curd has plentiful tastes and nice flavor with fine texture and rich nutrition. It is one of Chinese traditional condiments. Only in Taiwan, the total production in 1982 was about 12, 000 tons, i.e. 660 grams per person for a year (Shen De-zhong, Oversea Production and Study of sufu, Chinese Condiment, Issue 7, 1989, pages 2-4 and 26). So, there is a great demand for sufu, especially for

20 Chinese people over the world.

How to efficiently manufacture sufu but keep its good taste is one of the important topics.

Normally it takes 6-10 months to produce sufu from beans by using the existing methods, 25 which increases the risk of semi-products being contaminated by undesired

microorganisms.

The traditional preparation method takes soybean as raw material and makes massive bean curd by coagulation, and then inoculates the bean curd with mould to perform

30 fermentation, and forms the cube-like food. The main deficiency is the long time for

fermentation that makes the product easy to be decomposed and deteriorated. The Chinese patent application publication CN1357250 A discloses a method for making cube-shaped fermented bean curd. The disclosed method produces fermented bean curd, and it has the following features: placing the cut small cubes of bean curd into a fermentation room subjected to a pre-fermentation for 4 to 5 days to make the small cubes of bean curds ripened by showing little hyphae on their surface and emitting fragrance; and then, adding seasoning agents into the fermented and ripened small cubes of bean curds, and bottling and sealing the seasoned, fermented and ripened small cubes of been curds; and finally, giving a post-fermentation for 30-90 days to form the final products.

The Chinese patent application publication CN101396099A discloses a method for making a preserved bean curd, comprising inoculating at a temperature below 40 ^°C and pre-fermenting for 36 to 48 hours; and pickling for 3-5 days and post-fermenting for 45- 60 days at a temperature in the range of 15 to 30 ^°C .

Therefore no matter which method is used to produce sufu, one key problem that influences the efficiency of the fermentation process and the quality of final products exits in the preparation process is the fermentation period is overlong and the bean curd billets or blanks are easy to be rotten and putrefied during the fermentation.

The CN101283772A discloses a method for quickly preparing low-salt preserved bean curd by fermentation method, said method comprising the following steps: (1) sterilizing the bean curd prepared from the soybean material and cooling to room temperature; (2) inoculating mold and fermenting for 24 to 48 hours; (3) adding an enzyme preparation to promote maturation, fermenting for 5 to 7 days; and (4) blending, properly packaging and sterilizing after maturation of the preserved bean curd to obtain low-salt preserved bean curd paste or pieces. The present inventors studied the process for producing sufu, and identified the substance as ripening-promoting agent. Further, the present inventors provided a method for quickly manufacturing sufu.

Summary of the invention

According to the present invention, accelerated sufu ripening can be obtained by the use of carboxypeptidases. The present application provides ripening-promoting agent for sufu. The ripening-promoting agent can be a protease (preferable a carboxypeptidase preparation), or a lipase, or a combination of a protease (preferable a carboxypeptidase preparation) and a lipase. The ripening-promoting agent can be mixture of two or more proteases (preferable a carboxypeptidase preparation), or of two or more lipases, or of a combination of two or more proteases and two or more lipases. In a preferred

embodiment, the ripening-promoting agent is a combination of protease (preferable a carboxypeptidase preparation) and lipase. In a most preferred embodiment, the ripening- promoting agent is a combination of Accelerzyme CPG and Piccantase A.

And, the present application provides a method for quickly manufacturing sufu. The method is characterized in that a combination of protease (preferable a carboxypeptidase preparation) and lipase is added.

The present invention provides a method of preparing preserved bean curd wherein at least one of the protease is added into soybean milk and it is inoculated with mould after the soybean milk forms curd. Preferably at least one of lipase is added into the soybean milk. The protease is preferably an exo-proteases, more preferably a carboxypeptidase, even more preferably a Accelerzyme CPG.

In another embodiment, at least one lipase is added into the lipase is from microbial sources. Preferably, the lipase is Piccantase A. In another embodiment, the lactic acid bacteria are used to form curd from soybean milk. Preferably, the lactic acid bacteria is cultures selected from group consisting of

Lactococcus lactis ssp cremoris, Lactococcus lactis ssp lactis, Lactobacillus delbrueckii ssp bulgaricus, Streptococcus lactis, Streptococcus cremoris, Leuconostoc citrovor m, Streptococcus diacetilactis, or a mixture thereof. Preferably, the lactic acid bacteria is selected from DELVO-TEC® LL50 or DELVO-YOG® SVV series.

The mould comprises a single preserved bean curd production strain or mixtures thereof. The preserved bean curd production strains include Actinomucor elegans, Wutongqiao Mucor, Racemosus Mucor, Preserved bean curd Mucor.

The soybean raw material is a single product or mixture selected from group of soybean, soybean powder, bean meal, bean dreg, and bean cake. The present invention further provides the use of protease (preferable a carboxypeptidase preparation) in preparing preserved bean curd, wherein at least one protease is added into soybean milk before the mould is inoculated. Preferably the protease is Accelerzyme CPG®. At least one lipase is added into soybean milk before the mould is inoculated. Normally the lipase is from a microbial source. Preferably, the lipase is Piccantase® A.

The present invention further relates to a preserved bean curd prepared by the method according to present invention.

Any kinds of packaging method can be used based on the knowledge from a person in this art, the packaging method preferably comprises glass jar, PVDC (PVC), or plastic box. Detailed description of the invention

In one aspect, the present invention is to provide one or more enzyme additives for making sufu. The additive can be a single substance or a combination of two or more substances. The additive works as ripening-promoting agent.

In another aspect, the present invention relates to the use of protease in a method of preparing preserved bean curd. The purpose of this invention is to overcome the shortcomings of existing technology to provide a quicker ripening production process compared to the traditional method of preserved bean curd.

Additionally, the invention not only provides a time-saving process for preserved bean curd industrial production, but also gives the better quality of preserved bean curd.

Protease includes both endo -proteases and exo -pro teases.

Many proteases are involved in the generation of preserved bean curd flavours.

The generation of the proper flavour for a preserved bean curd requires a delicate balance of proteolytic activities from the proteases involved. Any imbalance will easily lead to flavours that are not wanted, such as-bitterness development. The development of proteases for preserved bean curd to enhance ripening processes is therefore a very delicate and complicated process. But, if the other materials or steps have produced some proteases, only a single protease may be added. Exo-proteases are preferred over endo- proteases because they have a lower tendency to induce formation of bitterness.

Over the years, several commercial protease preparations have been introduced into the market. An overview of available commercial products is given in several papers (Wilkinson, van den Berg & Law, Bulletin of the IDF (2002) 371,16-19 ; Kilcawley, Wilkinson & Fox, Food Biotechnol (2002) 16,29-55 ; Kilcawley, Wilkinson & Fox, Enzyme Microb.

Technol. (2002) 31,310-320). Examples include enzyme preparations derived from fungal species (including but not limited to Bioprotease P Cone and Bioprotease A cone from Quest, The Netherlands, Protease M & Protease A and Acid protease A from Amano, Promod 215 from Biocatalysts, Sternzyme B5026 from Stern, Flavourzyme MG/A from Novozymes, Denmark) and bacterial species (including but not limited to Protamex and Neutrase from Novozymes, Denmark, Protease N from Amano, Promod 24P and. 24L from Biocatalysts, Protease B500 from DSM, The Netherlands, and Protease 200L from Rhodia Foods, France). The proteases vary considerably in composition with respect to presence of specific proteases and/or the ratio in which these proteases occur in a specific product. The papers by Kilcawley, Wilkinson and Fox, referred to above, clearly show that most commercial protease products are a mixture of endo-and exo-peptidase activities. Several products are developed to contain only exo- peptidase activity. For food applications, these are invariably amino-peptidases, and examples include DBS50 and DBP20 (from Rhodia, France), Corolase LAP (from Rohm, Germany), Flavourzyme MG/A (from Novozymes, Denmark), Accellerzyme AP and Accelerzyme ® CPG (from DSM, The Netherlands) and Peptidase R (from Amano, Japan).

In this invention, an exo-proteases is preferred. In a more preferred embodiment, the carboxypeptidase preparation is used. In a most preferred embodiment, the

carboxypeptidase is Accelerzyme ® CPG.

Accelerzyme ® CPG is a purified enzyme obtained by submerged culturing of a selected strain of Aspergillus niger. The enzyme has a broad spectrum activity for release of C- terminal amino acids from the proteins and peptides. The disclosure of it in the PCT publications, WO 2005/074695 and WO 2009/080484, is incorporated into this patent application by reference. The enzyme or enzyme mixture having the similar activity to Accelerzyme ® CPG can be used in the present invention. For example, the enzyme Accelerzyme NP 50000 can also be used. The proteases preparation addition for preserved bean curd ripening can be done in various stages of preserved bean curd preparation before inoculating the mould.

Preferably, the protease is added to the soy bean milk. This may be prior to or together with the addition of the coagulant. Addition to the soy bean milk promotes a homogenous distribution of the enzymes throughout the preserved bean curd. After the soybean milk forms curd, the mould is inoculated.

According to the present invention, accelerated preserved bean curd can be obtained by the use of proteases. The proteases should be able to at least release amino acids that are important for preserved bean curd flavour formation, such as leucine, phenylalanine, valine and methionine. The protease is added at activity levels between 0.001wt% and lwt% substrate (e. g. soy bean milk), preferably 0.05-0.5wt% substrate or more preferably 0.01-0.1wt% substrate.

The carboxypeptidase preparation addition for sufu ripening can be done in various stages of sufu preparation. Preferably, the carboxypeptidase preparation is added to the soybean milk. This may be prior to or together with the addition of the coagulant.

Addition to the soybean milk promotes a homogenous distribution of the enzymes throughout the sofu. Alternatively, the enzymes can be added at a later stage, but this introduces the risk of inhomogeneous enzyme distribution in the sofu.

In a preferred embodiment, the carboxypeptidase preparation is free from endoprotease activity, and is able to at least release amino acids that are important for sufu flavour formation, such as leucine, phenylalanine, valine and methionine.

The carboxypeptidase preparation is defined as free of endo-protease activity when the ratio of endo-protease activity (PU) / carboxypeptidase activity (CPG) in the preparation is less than 0.01, preferably less than 0.001 and most preferably less than 0.0005. The carboxypeptidase preparation preferably shows broad spectrum carboxypeptidase activity through which it is able to release the majority of the natural amino acids from peptides or proteins. Broad spectrum carboxypeptidase activity is defined as activity which releases at least 80% of the natural amino acids in amounts detectable by the method as described in example 3 of WO2005/074695.

In a preferred embodiment of the invention, the carboxypeptidase activity of the carboxypeptidase preparation is for at least 90% caused by a single enzyme, whereby carboxypeptidase activity is measured as described in example 1 of WO2005/074695. In another preferred embodiment, combinations of carboxypeptidases are used.

In a preferred embodiment of the invention, the carboxypeptidase activity is from a purified carboxypeptidase CPD I (PEPG) from Aspergillus niger.

In a third aspect, the present invention is about use of lipase in the manufacture of sufu.

In some embodiments, it may also be beneficial to use lipase such as rennet paste prepared from calf, lamb, kid stomachs, or Palatase A750L (Novo), Palatase M200L (Novo), Palatase M1000 (Novo), or Piccantase A (DSM), also Piccantase from animal sources from DSM (K, KL, L & C) or Lipomod 187, Lipomod 338 (Biocatalysts). The use of these lipases may have a beneficial effect on flavour in the preserved bean curd. The lipase addition for preserved bean curd ripening can be done in various stages of preserved bean curd preparation before inoculating the mould. Preferably, the lipase is added to the soy bean milk. This may be prior to or together with the addition of the coagulant. Addition to the soy bean milk promotes a homogenous distribution of the enzymes throughout the preserved bean curd. After the soybean milk forms curd, the mould is inoculated.

At least one of lipase is added into the soybean milk. Preferably, the lipase is from microbial sources. More preferably, the lipase is Piccantase ® A. Piccantase ® A is a microbial lipase preparation derived from the fungus Rhizomucor miehei.

Similar enzyme or enzyme preparation can also be used. In particular, the following lipase enzymes can be used:

Piccantase R800;

Piccantase AN;

Piccantase K;

Piccantase L;

Piccantase KL;

Piccantase L-5X; and

Piccantase C-3X.

All the above lipases can be commercially obtained from DSM, the Netherlands.

The lipase is added at activity levels between 0.001wt% and lwt% substrate (e. g. soy bean milk), preferably 0.05-0.5wt% substrate or more preferably 0.01-0. lwt% substrate.

At least one of cultures of lactic acid bacteria is used as coagulant to form curd from soybean milk. Examples of the normal cultures are selected from cultures used for the preparation of fresh cheese consisting of Lactococcus lactis ssp cremoris, Lactococcus lactis ssp lactis, Lactobacillus delbrueckii ssp bulgaricus, Streptococcus lactis,

Streptococcus cremoris, Leuconostoc citrovorum, Streptococcus diacetilactis, or a mixture thereof. In a preferred embodiment, cultures of lactic acid bacterium are selected from DELVO-TEC® LL50 or DELVO-YOG® SVV series.

At least one of mould which is selected from the group consistingAcrinomMcor elegans, Wutongqiao Mucor, Racemosus Mucor, Preserved bean curd Mucor, or mixture thereof will be used in the manufacture of sufu. The soybean raw material can be a single product or mixture selected from group of soybean, soybean powder, bean meal, bean dreg, and bean cake.

After the preserved bean curd prepared by the invention method, it can be packaged into a glass jar, PVDC (PVC), or plastic box.

In a preferred embodiment, the specific steps of the method for quickly producing sufu according to this invention are as follows:

(1) sterilize the soy-bean milk by boiling, and then cool it down to room temperature; (2) add in the order of protease, lipase, lactic acid bacteria series, and keep warm for 10 tol5 hours at 30 ^°C ;

(3) when the soybean milk forms curd having a smooth and tenacious texture, cut it into small pieces with 1.5cm in length, 1.5cm in width, and 1.5cm in high;

(4) inoculate with mould, and ferment for 48 hours at 28 ^°C ;

(5) inactivate enzyme, after hydrolysis for 6-10 hours at 45-50 ^°C ; and

(6) immerse the preserved bean curd into supplementary material, and when package mature, then you can get massive end product of preserved bean curd.

In a specific embodiment, the ripening-promoting agent, i.e. Accelerzyme CPG and Piccantase A is used in the method for making sufu in the shape of cube, the method comprises the following steps: (1). grind soybean to form a soybean milk, boil the soybean milk to sterilize it, and cool the sterilized soybean milk down to room

temperature; (2). add Accelerzyme CPG, Piccantase A, lactic acid bacteria LL50 or SVV into the sterilized and cooled soybean milk, and keep the milk at 30 "C for 10 to 15 hours; (3). When the soybean milk forms curd having a finely smooth and firm texture, cut it into small pieces of 1.5 cm X 1.5 cm X 1.5 cm; (4) inoculate mould onto the small pieces, and ferment the inoculated small pieces at 28 ^°C for 48 hours; (5) hydrolyze for 6-10 hours at 45-50 ^°C and then inactivate the enzymes; and (6). soak the sufu cubes into adjuvant, and package with suitable packaging way after repining so that the final products in cube shape are formed. In the method for quickly making sufu in the shape of cube by using the ripening- promoting agent, i.e. Accelerzyme CPG and Piccantase A, the raw materials may be soybean, soybean powder, soybean pulp, residue from beans after making soya-bean milk, or bean cake, and other articles made from soybean, and a mixture thereof.

The fermenting agent, as used, may be LL50 series, SVV series, and etc.

The mould, as used, may comprise a single strain or a mixture of strains as used in the traditional method for making sufu, including A ctinomucor elegans, Mucor wutong kiao, Mucor racemosus, Mucor sufu, and etc.

The package, as used, includes glass bottles, PVDC (PVC) plastic boxes, and etc.

The aim of the present invention is to overcome the disadvantages of the existing technologies, by providing a ripening-promoting agent for sufu. The ripening-promoting agent can be used in the manufacturing method for making sufu, including the traditional method. The use of the ripening-promoting agent shortens the production recycle, and helps the modernization of the manufacturing method for making sufu, and imparts good quality to the cube-shaped sufu.

The advantages and good effects of the present invention include:

1. The present invention uses ripening-promoting agent, for example, protease and lipase, to promote the ripening of sufu, which shortens the production cycle.

2. The present invention provides a process for using the ripening-promoting agent, for example, protease and lipase, in the method for making sufu, as well as the method for making sufu by the ripening-promoting agent. This contribution improves the quality of cube-shaped sufu, and provides a basis for modernized production of cube-shaped sufu in a quick way. The following illustrative examples are set forth to provide a clearer understanding of the invention and are not to be construed as limiting the principles which underlie the spirit and scope of the invention. The examples are provided herewith only for the purpose of description, not to limit the scope of protection of the present invention.

Now, typical examples are used to specifically describe the invention. Examples Example 1

Take 10.0kg of dry soybean as the raw material, and add 25kg of water into it. Soak the dry soybean in the water for 10 hours, and then pour the soybean and water into a grinder. Into the grinder, adding 60.0kg of additional water and grind the soybean into the milk status by wet milling. Sterilizing the soybean milk by heating it up to boiling, and then cooling it down to room temperature;

Into the above sterilized and cooled soybean milk, add in the order of 0.01% of Accelerzyme® CPG (commercially available from DSM, the Netherlands), 0.02% of Piccantase® A (commercially available from DSM, the Netherlands), 0.01% of DELVO- TEC® LL50-A (commercially available from DSM, the Netherlands, the composition of the culture is Lactococcus lactis ssp cremoris and/or Lactococcus lactis ssp lactis), and 0.02% of CaCl2, and keep the mixture warm for 12 hours at 30 ^°C ;

After the soybean milk has formed curds having a smooth and tenacious texture, the curds are cut into small pieces with the specification of 1.5cm in length, 1.5cm in width, and 1.5cm in height;

Inoculate the pieces with Actinomucor elegans, which is one strain for fermentation to producing bean curds, and ferment the inoculated pieces of bean curd for 48 hours;

Inactivate the enzymes after a constant-temperature fermentation for 8 hours at 50 ^°C; and

Immerse the fermented bean curd into a suspension comprising 4% of salt, 3% of yellow rice wine and 0.01% of spice powder, and package them into glass jars after the fermented bean curd gets matured, then form the massive end-product of fermented bean curd.

Example 2:

Add 10kg of whole soybean powder as raw material into 100kg of water so that the whole soybean powder dissolves in the water completely. The solution obtained was boiled for 3 minutes to form soybean milk, which was then naturally cooled down to the room temperature;

Into the above solution as obtained in step , add in the order of 0.05% of

Accelerzyme® CPG(DSM), 0.02% of Piccantase® A(DSM), 0.01% and 0.01% of DELVO-YOG® SVV-21(DSM) and CaCl₂. Then, the mixture was kept warm for 8 hours at 30 V ;

When the soybean milk forms curd having a smooth and tenacious texture, cut the curd into small pieces with 1.5cm in length, 1.5cm in width, and 1.5cm in high;

Inoculate the small pieces with the strain Actinomucor elegans for the production of fermented bean curd, and ferment the inoculated small pieces for 48 hours;

Inactivate the enzymes, after the constant-temperature fermentation for 8 hours at 50 °C on the small pieces;

Immerse the fermented bean curd into a suspension comprising 4% of table salt, 3% of yellow rice wine and 0.01% of spice powder, and package them into plastic barrels when the bean curd get matured, then the end product of fermented bean curd in cube shape was obtained.

Example 3:

Fully dissolve 20kg of soybean meal as raw material into 150kg of water, boil the mixture for 5 minutes. Soy milk was obtained by passing the mixture through 80 mesh sieve. Then the soy milk was naturally cooled down to room temperature;

Into the soy milk as obtained in , add in the order of 0.05% of Accelerzyme® CPG(DSM), 0.03% of Piccantase® A(DSM), 0.01% of DELVO-YOG® SVV-21(DSM, a culture of Lactobacillus delbrueckii ssp bulgaric s, commercially available), and 0.02% of CaCl₂, and keep the so-obtained mixture warm for 9 hours at 30 ^°C ;

Inoculate the small pieces of curd with Actinomucor elegans, the strain for the production for fermented bean curd, and ferment the inoculated small pieces of curd for 48 hours;

Inactivate the enzymes, after the constant-temperature fermentation for 10 hours at 45

^°C ; and

Immerse the fermented bean curd into the suspension comprising 4% of table salt, 3% of yellow rice wine and 0.01% of spice powder for ripening the fermented bean curd. After the bean curd has got matured, it was packaged into glass jars. Thereby, the end product of fermented bean curd in cube shape was obtained.

Claims

1. A method of preparing preserved bean curd, characterizing in that at least one carboxypeptidase preparation is used.

2. The method of claim 1, wherein the carboxypeptidase is added to the soybean milk.

3. The method of claim 1 or 2, wherein the ration of endoprotease activity and carboxypeptidase activity in the carboxypeptidase preparation is less than 0.01, preferably less than 0.001, and most preferably less than 0.0005.

4. The method of any of proceeding claims, wherein at least one lipase is added.

5. The method of any of proceeding claims, wherein the lipase can be added before, simultaneously or after the adding of carboxypeptidase preparation.

6. The method of claim 4, wherein the lipase is from microbial sources.

7. The method of claim 6, wherein the lipase is a lipase preparation derived from the fungus Rhizomucor miehei.

8. The method of any of the claim 1 - 7, further comprising the use of lactic acid bacteria.

9. The method of claim 8, wherein lactic acid bacteria is cultures elected from group consisting of Lactococcus lactis ssp cremoris, Lactococcus lactis ssp lactis, Lactobacillus delbrueckii ssp bulgaricus, Streptococcus lactis, Streptococcus cremoris, Leuconostoc citrovorum, Streptococcus diacetilactis, or the mixture thereof.

10. The method of claim 8, wherein lactic acid bacteria is selected from the composition of the culture of Lactococcus lactis ssp cremoris and Lactococcus lactis ssp lactis or the composition of the culture of Lactobacillus delbrueckii ssp bulgaricus and Streptococcus diacetilactis .

11, The method of claim 1, wherein the mould comprises a single preserved bean curd production strain or mixtures thereof, and said preserved bean curd production strain is selected from the group consisting of Actinomucor elegans, Wutongqiao Mucor, Racemosus Mucor, Preserved bean curd Mucor, or mixture thereof.

12. The method of claim 1, wherein the soybean raw material is selected from the groups consisting of soybean, soybean powder, bean meal, bean dreg, and bean cake or the mixture thereof.

13 Use of carboxypeptidase preparation in preparing preserved bean curd.

14. The use of claim 13, wherein the ration of endoprotease activity and carboxypeptidase activity in the carboxypeptidase preparation is less than 0.01, preferably less than 0.001, and most preferably less than 0.0005.

15. The use of claim 13 or 14, wherein, at least one lipase is used.

16. The use of claim 15, wherein the lipase can be added before, simultaneously or after the adding of carboxypeptidase preparation.

17. The use of claim 15, wherein the lipase is from microbial sources.

18. The use of claim 17, wherein the lipase is a lipase preparation derived from the fungus Rhizomucor miehei.

19. A preserved bean curd which is prepared by the method according to any of claims 1 to 13.