CN111635870A

CN111635870A - Leavening agent, direct-feeding fermentation method of stinky tofu marinating liquid and stinky tofu marinating liquid

Info

Publication number: CN111635870A
Application number: CN202010455797.XA
Authority: CN
Inventors: 谢静莉; 魏东芝; 李立; 邓思敬; 沈建华; 王家军; 马新新; 赵振阳; 沈沪铭
Original assignee: Shanghai Tramy Green Food Group Co ltd; East China University of Science and Technology
Current assignee: Shanghai Tramy Green Food Group Co ltd; East China University of Science and Technology
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2020-09-08
Anticipated expiration: 2040-05-26
Also published as: CN111635870B

Abstract

The invention discloses a leavening agent, a direct-vat-set fermentation method of stinky tofu marinating liquid and the stinky tofu marinating liquid. The leaven is compounded by 4 strains of lactic acid bacteria. The direct vat set fermentation method of the strong-smelling preserved bean curd comprises the following steps: inoculating a starter in the stock solution of the halogen solution, wherein the initial total concentration of the starter in the stock solution of the halogen solution is 10^3‑6CFU/mL, culturing at 30-45 ℃ for 10-15 h, then adding a fermentation nitrogen source according to the volume ratio of 1-5%, and continuing to ferment for 10-15 h to obtain the stinky tofu marinating liquid. The method comprises mixing and fermenting the stock solution of bittern with 4 kinds of specific lactobacillus, mixing the raw materials and lactobacillusThe fermented bean curd marinating liquid with uniform quality and flavor can be obtained by directly feeding the raw materials according to the proportion and fermenting under specific fermentation conditions, and the fermentation period is short.

Description

Leavening agent, direct-feeding fermentation method of stinky tofu marinating liquid and stinky tofu marinating liquid

Technical Field

The invention belongs to the technical field of fermented bittern of strong-smelling preserved bean curd. In particular to a leavening agent, a direct vat set fermentation method of a stinky tofu marinating liquid and the stinky tofu marinating liquid.

Background

The strong-smelling preserved bean curd is a traditional food of Chinese nationality, has unique flavor and nutritive value, contains rich essential amino acids for human bodies and VB12 and has unique biological activity. Due to different dietary habits all over the country, the preparation process and the aroma components of the strong-smelling preserved bean curd are greatly different. The traditional strong-smelling preserved bean curd is prepared by naturally fermenting vegetables and spices serving as raw materials according to a certain proportion. The formula of the strong-smelling preserved bean curd marinating liquid in the Hunan area is mainly prepared by naturally fermenting raw materials of fermented soya beans, calcined soda, blue vitriol, mushrooms, winter bamboo shoots and the like, and the surface of the soaked strong-smelling preserved bean curd is black; the formula of the stinky tofu marinating liquid in the areas represented by Shaoxing in south China mainly comprises amaranth stems, ginger, pepper and other raw materials, and the surface of the brewed tofu is in a beige color.

During the fermentation process of the stinky tofu marinating liquid, microorganisms play an important role, and the types and the content of the microorganisms play a decisive role in the formation of the flavor and the improvement of the nutritional value of the stinky tofu marinating liquid. The stinky tofu brine solution has high relative content of volatile aroma components such as alcohols, ketones, esters, aldehydes and the like (congratulation, thank you, Liu army pigeon and the like. the change trend analysis of the volatile components in the stinky tofu brine fermentation process [ J ]. Chinese fermentation, 2016,35(7):79-84.), and alkanes, alcohols, aldehydes, esters and acids (Zheng Xiao fen, Su Wu, Jiangli. comparison of the volatile components in two stinky tofu brines [ J ]. Chinese brewing, 2013,32(10):122 and 125.). Wherein, the n-butyl alcohol has mellow fragrance, the eucalyptus mellow fragrance is characterized by camphor odor, the linalool is characterized by muguet odor, the 4-terpene alcohol fragrance is characterized by wood and soil odor, and the fragrances can endow the strong fragrance of the stinky tofu halogen liquid and form important fragrance characteristics of the halogen liquid (Zhuruihong, Xuehu Cheng, Lichen, and the like. The above alcohols are also present in soy sauce, fermented bean curd, etc. (LEE S J, AHN B. Complex of calcium compositions in fermented soybean processing and extraction (SDE) with sensorial microorganisms [ C ]. fourteenth world food science and technology Association, Shanghai: Chinese food science and technology society, 2008: 10-19.). Liuyuping, Miao Shiwei, Huangming quan, and the like volatile aroma components in the stinky tofu are extracted and separated [ J ]. Food science, 2011,32(24): 228-.

However, in the prior art, the traditional method for industrially producing the strong-smelling preserved bean curd marinade is usually formed by natural fermentation in an open jar or a cement pool. The microorganism used as the leaven comes from natural environment such as air, the type of the microorganism is difficult to determine, and harmful microorganism enters the microorganism to bring food safety hidden trouble. The traditional method for industrially producing the strong-smelling preserved bean curd marinating liquid is half a year to 1 year, then the strong-smelling preserved bean curd is repeatedly soaked, the soaking time can be as long as 2 years or even longer, and various microorganisms and mixed bacteria can be added in the process, so that the product quality is unstable. Because the fermentation process is simple and extensive, the mechanization degree is low, the production period is long, and the time, the temperature, the raw material proportion and the like lack clear production standards, the strong-smelling preserved bean curd marinating liquid with clear microorganism types and stable flavor can not be formed, the quality of the strong-smelling preserved bean curd is difficult to control, and the development of related industries is seriously hindered.

Therefore, the direct vat set fermentation method of the strong-smelling preserved bean curd with the fixed formula is developed, and the method has very important significance for improving the industrial production level of the strong-smelling preserved bean curd and the strong-smelling preserved bean curd products with stable production quality, safety and flavor.

Disclosure of Invention

The invention aims to provide a direct vat set lactic acid bacteria starter of a stinky tofu marinating solution and a direct vat set fermentation method of the stinky tofu marinating solution, wherein the specific 4 lactobacillus strains are used for mixing and fermenting the marinating solution stock solution to obtain the stinky tofu marinating solution with stable quality, safety and flavor, the fermentation time of the stinky tofu marinating solution is short, the production time of the stinky tofu is short, and therefore the industrial production of the stinky tofu can be realized.

In order to achieve the purpose, the invention adopts the following technical scheme:

a leaven, wherein the leaven is compounded by 4 strains of lactic acid bacteria; wherein:

4 strains of the lactic acid bacteria are Lactobacillus plantarum KS1-1, Lactobacillus plantarum KS1-2, Pediococcus pentosaceus ZJL2-2 and lactococcus lactis ZJL1-3 respectively; the lactobacillus plantarum KS1-1 has a deposit number of: CCTCC NO: M2020131; the lactobacillus plantarum KS1-2 has a deposit number of: CCTCC NO: M2020132; the preservation number of the pediococcus pentosaceus ZJL2-2 is as follows: CCTCC NO: M2020133; the lactic acid galactococcus ZJL1-3 has the preservation number: CCTCC NO: M2020134.

As is readily understood by those skilled in the art, the 4 strains of lactic acid bacteria can be compounded according to a certain proportion.

Preferably, the leavening agent is 4 strains of lactobacillus which are compounded in equal amount.

As will be readily understood by those skilled in the art, the 4 strains of lactic acid bacteria can be stored in a certain manner.

Preferably, the lactic acid bacteria are freeze-dried lactic acid bacteria powder.

In the invention, the leaven can be used for fermenting the conventional bittern stock solution for preparing the stinky tofu bittern of the prior art.

Preferably, the components of the stock solution of the marinade comprise amaranth stems, ginger, pepper and sterile saline; in the brine stock solution, the concentration of the amaranth stalks is 10-50 g/100mL, the concentration of the ginger is 1-10 g/100mL, the concentration of the pepper is 0.5-5 g/100mL, and the concentration of the sterile saline water is 1-5 g/100 mL.

Preferably, the compound ratio range of the lactobacillus plantarum KS1-1, the lactobacillus plantarum KS1-2, the pediococcus pentosaceus ZJL2-2 and the lactococcus lactis ZJL1-3 is as follows: 1 (0.1-3), (0.5-5) and (0.1-5).

More preferably, the compounding ratio range of the lactobacillus plantarum KS1-1, the lactobacillus plantarum KS1-2, the pediococcus pentosaceus ZJL2-2 and the lactococcus lactis ZJL1-3 is as follows: 1 (0.5-2), (1-3) and (0.5-4).

The second purpose of the invention is to provide a direct-vat-set fermentation method of the stinky tofu marinating liquid, which comprises the following steps:

inoculating the leavening agent into a halogen liquid stock solution, wherein the initial total concentration of the leavening agent in the halogen liquid stock solution is 10^3-6CFU/mL, culturing at 30-45 ℃ for 10-15 h, then adding a fermentation nitrogen source according to the volume ratio of 1-5%, and continuing to ferment for 10-15 h to obtain the stinky tofu marinating liquid.

According to a preferred technical scheme of the invention, the 4 strains of lactic acid bacteria are equivalently compounded in the form of freeze-dried bacteria powder to be used as the leavening agent.

More preferably, in the stock solution of the marinating solution, the concentration of the amaranth stalks is 12.25g/100mL, the concentration of the ginger is 1.25g/100mL, and the concentration of the pepper is 0.63g/100 mL;the concentration of the sterile saline is 2g/100 mL; the initial total concentration of the leavening agent in the stock solution of the brine is 10⁴CFU/mL。

Under the formula conditions, the microbial contamination can be effectively inhibited, the fermentation effect of the lactic acid bacteria is best, and the microbial concentration at the fermentation end point is highest.

Preferably, the stock solution of the fermented bean curd is cultured at 37 ℃ for 12h, then a fermentation nitrogen source is added according to the volume ratio of 2%, and the fermentation is continued for 12h to obtain the fermented bean curd halogen solution.

Under the fermentation conditions, the strong-smelling preserved bean curd marinating liquid with good fermentation effect can be obtained in a short time.

Further preferably, the fermentation nitrogen source is a ground bean curd base. Of course, the fermentation nitrogen source can also be a nitrogen source which is conventionally used in the fermentation of the fermented bean curd marinade in the prior art, such as soybean milk.

The third purpose of the invention is to provide the stinky tofu marinating liquid which is obtained by adopting the direct-vat fermentation method of the stinky tofu marinating liquid.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) the starter of the invention can be used as a starter to be directly put into a halogen liquid raw material by compounding the specific 4 kinds of lactic acid bacteria, the fermenting capability of the starter after the 4 strains of lactic acid bacteria are compounded is obviously improved compared with that of the starter which is independently used as the lactic acid bacteria, and the bacterial concentration at the fermentation end point is obviously higher than that of the single strain of lactic acid bacteria. Therefore, the fermented bean curd marinating liquid with good fermentation effect is obtained by fermenting the marinating liquid raw material under the industrial production condition, the fermentation time can be shortened to 1 day, and compared with the prior art, the fermentation time is obviously shortened. Therefore, the method is suitable for industrial large-scale production.

(2) According to the direct vat set fermentation method of the stinky tofu marinade, the marinade stock solution is fermented by compounding the specific 4 lactobacillus strains serving as a leavening agent, the leavening strains are clear, and the fermentation conditions such as time, temperature, raw material ratio and the like are clear; the raw materials and the lactic acid bacteria are directly added according to the proportion and fermented under the specific fermentation condition, so that the strong-smelling preserved bean curd marinating liquid with stable quality, safety and flavor can be obtained, and the fermentation effect is good; and the fermentation time of the fermented bean curd marinating liquid and the soaking production period of the fermented bean curd are obviously shortened, so that the industrial production of the fermented bean curd can be realized.

(3) The lactobacillus plantarum KS1-1 and the lactococcus lactis ZJL1-3 in the lactobacillus fermentation strain have obvious bacteriostatic action on common pathogenic bacteria, and the food safety risk in the production process of the strong-smelling preserved bean curd can be greatly reduced.

Drawings

FIG. 1 is a graph showing the change of the concentration of the fermented bean curd halogen liquid during the fermentation of a single strain of Lactobacillus plantarum, lactococcus lactis and Pediococcus pentosaceus.

FIG. 2 is a graph showing the change of the concentration of the fermented bean curd halogen liquid with time during the complex fermentation of 4 strains.

FIG. 3 is a graph showing the pH/acidity-time change of the fermented bean curd halogen solution during the complex fermentation of 4 strains.

FIG. 4 is a graph showing the color-time change of the fermented bean curd halogen liquid during the complex fermentation of 4 strains.

FIG. 5 is a GC-MS spectrum of the volatile aroma components of the unfermented marinade.

FIG. 6 is a GC-MS spectrum of volatile aroma components in fermented strong-smelling preserved bean curd marinating liquid.

Deposit description

The Lactobacillus plantarum KS1-1(Lactobacillus plantarum KS1-1) strain disclosed by the invention is stored in China Center for Type Culture Collection (CCTCC for short) in 5-19 th of 2020, and the address of a storage unit is as follows: china, Wuhan university, the preservation number is CCTCC NO: M2020131.

The Lactobacillus plantarum KS1-2(Lactobacillus plantarum KS1-2) strain disclosed by the invention is stored in China Center for Type Culture Collection (CCTCC for short) in 5-19 th of 2020, and the address of a storage unit is as follows: china, Wuhan university, the preservation number is CCTCC NO: M2020132.

The Pediococcus pentosaceus ZJL2-2(Pediococcus pentosaceus ZJL2-2) strain is stored in China Center for Type Culture Collection (CCTCC) in 5 months and 19 days of 2020, and the storage unit address is as follows: china, Wuhan university, the preservation number is CCTCC NO: M2020133.

The Lactococcus lactis ZJL1-3(Lactococcus lactis ZJL1-3) strain of the invention is stored in China Center for Type Culture Collection (CCTCC for short) in 5 months and 19 days of 2020, and the storage unit address is as follows: china, Wuhan university, the preservation number is CCTCC NO: M2020134.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The 4 strains of lactic acid bacteria used for fermentation in the invention are all separated from pickle and sour dough and are lactobacillus plantarum KS1-1, lactobacillus plantarum KS1-2, pediococcus pentosaceus ZJL2-2 and lactococcus lactis ZJL1-3 respectively.

MRS liquid medium: 10.0g of peptone, 5.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 1.0mL of Tween-80, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 2.0g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 1000mL of distilled water.

MRS solid medium: 10.0g peptone, 5.0g beef powder, 4.0g yeast powder, 20.0g glucose, 1.0mL tween-80, 2.0g dipotassium hydrogen phosphate, 5.0g sodium acetate, 2.0g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 16.0g agar powder and 1000mL distilled water.

LB liquid medium: 10g of NaCl, 10g of tryptone, 5g of yeast extract and 1000mL of distilled water.

LB solid medium: 10g of NaCl, 10g of tryptone, 5g of yeast extract, 16.0g of agar powder and 1000mL of distilled water.

The raw materials and equipment used in the invention are all commercially available.

Example 1 isolation and identification of lactic acid bacteria

The lactic acid bacteria strain is separated and identified from naturally fermented pickle and sour dough, and the method comprises the following steps:

(1) diluting the collected sauerkraut and sour dough with sterile normal saline 10 times, respectively diluting to 10^-3And 10^-5And (2) taking 200 mu L of the diluent, uniformly coating the diluent on an MRS solid culture medium (MRS culture medium mixture 48.3g/L containing 2% agar and sterilized at 115 ℃), carrying out inverted culture in a 37 ℃ constant temperature incubator for 48h, taking out, and then picking out a single colony to be cultured in 10mL of MRS liquid culture medium (containing 2% agar) for 18h in a 37 ℃ constant temperature shaking table at the rotating speed of 200 rpm.

(2) The obtained bacterial solution was subjected to polymerase chain reaction on 16S rRNA fragments after DNA extraction with a DNA extraction kit, wherein the primers are shown in Table 1, the reaction system is shown in Table 2, and the reaction conditions are shown in Table 3.

(3) And (3) sending the amplified product to Parsenno biotechnology, Limited liability company for sequencing, and comparing the sequencing result with the sequence of a standard strain on Genebank.

TABLE 1, 16S rRNA primer sequences

TABLE 2 amplification reaction System

TABLE 3 amplification reaction conditions

Sequencing and identifying the 16S rRNA amplification product sequence of the strain, comparing 4 strains with the standard strain sequence on GenBank to obtain the strain with the highest homology (> 99%), and determining the separated strains to be lactobacillus plantarum, pediococcus pentosaceus and lactococcus lactis respectively. The 4 strains were named as follows according to the identification results: lactobacillus plantarum KS1-1, Lactobacillus plantarum KS1-2, lactococcus lactis ZJL1-3, and Pediococcus pentosaceus ZJL2-2, as shown in Table 4.

TABLE 416S rDNA sequencing results comparison Table

Wherein KS1-1 and KS1-2 are isolated from sauerkraut, and ZJL2-2 and ZJL1-3 are isolated from sour dough.

Example 2 preparation of lyophilized powder of lactic acid bacteria

In an aseptic operation table, a small amount of bacterial liquid is picked from a glycerol tube for storing lactobacillus plantarum KS1-1, lactobacillus plantarum KS1-2, lactobacillus lactis ZJL1-3 and pediococcus pentosaceus ZJL2-2, streaked on an MRS solid plate, cultured at 37 ℃ for 36-48 h, a single colony on the picked plate is inoculated in 10mL of liquid MRS culture medium, cultured anaerobically at 37 ℃ for 18h and then inoculated in a 10L fermentation tank containing the MRS culture medium, fermented anaerobically for 24h, then centrifuged at 6000rmp for 10min to collect thalli, and washed 2 times by using sterile phosphate buffer (pH 6.4). And centrifuging, and fully mixing and emulsifying the obtained bacterial sludge and a sterilized freeze-drying protective agent containing 18-22% of skimmed milk powder, 4-9% of trehalose, 2-4% of sodium glycinate, 1-5% of glycerol and 0.1-1% (w/w) of cysteine hydrochloride according to a weight ratio of 1: 2. And after the bacterial sludge and the protective agent are fully and uniformly suspended, pre-freezing the emulsified mixed solution for 4-10 hours at the temperature of-45 ℃, and then putting the emulsified mixed solution into a freeze dryer for freeze drying for 20-26 hours to obtain freeze-dried bacterial powder. The viable count of Lactobacillus plantarum KS1-1, Lactobacillus plantarum KS1-2, lactococcus lactis ZJL1-3 and Pediococcus pentosaceus ZJL2-2 in the bacterial powder is 10^10～12CFU/g。

Examples 3-6 and 4 lactic acid bacteria fermentation of fermented Bean curd marinade alone

The method is characterized in that 4 lactic acid bacteria are independently used as a leavening agent for fermenting the stock solution of the brine, and the method comprises the following specific steps:

(1) preparing a stock solution of the brine solution, wherein the stock solution of the brine solution comprises amaranth stalks, ginger, pepper and sterile saline water, the concentration of the amaranth stalks is 12.25g/100mL, the concentration of the ginger is 1.25g/100mL, the concentration of the pepper is 0.63g/100mL, and the concentration of the sterile saline water is 2g/100 mL.

(2) Inoculating a starter in the halogen liquor stock solution of the step (1), wherein the initial total concentration of the starter in the halogen liquor stock solution is about 10⁴CFU/mL, culturing at 37 deg.C for 12 hr, adding 2% of ground bean curd blank as fermentation nitrogen source, and continuing to ferment at 37 deg.CAnd (5) obtaining the fermented bean curd marinating liquid after 12 hours.

The results of detecting the change of the concentration of the bacteria in the fermentation process of the step (2) are shown in FIG. 1.

As can be seen from figure 1, the lactobacillus plantarum KS1-1 and KS1-2 have good fermentation capacity, are exponentially increased in the early stage (0-12 h) of fermentation, grow slowly for about 12-18 h and enter a stable stage, and the bacterial concentration is 10 when the fermentation reaches 18h¹²～10¹³CFU/mL, after which the germ concentration is no longer changed, is primarily the flavor generation stage.

Pediococcus pentosaceus ZJL2-2 and lactococcus lactis ZJL1-3 also have good fermentation capacity, and grow exponentially in the early stage of fermentation (0-18 h), and grow slowly in about 18h and enter a stable stage, and the bacterial concentration is 10 when the fermentation reaches 18h⁸～10¹⁰CFU/mL, after which the germ concentration is no longer changed, is primarily the flavor generation stage.

Example 7 measurement of bacteriostatic ability of Single-Strain fermentation broth

The fermented bean curd marinating liquid obtained by fermenting the single strain in the embodiment 3-6 is used for bacteriostasis experiments, and the specific steps are as follows:

respectively activating Escherichia coli and Staphylococcus aureus in LB liquid culture medium at 37 deg.C overnight in a constant temperature shaker. Then, the LB liquid culture medium containing the activated escherichia coli or staphylococcus aureus is evenly added into the melted LB solid culture medium according to the proportion of 1 muL/mL by a pouring method, 5 oxford cups are respectively placed on the surface of the LB solid culture medium after the solid culture medium is in a semi-solidified state, then 100 muL of the stinky tofu halogen solution prepared in the embodiment 3-6 is respectively injected into the oxford cups, the stock solution of the halogen solution without the stinky tofu halogen solution is used as a reference, the culture is carried out at the constant temperature of 37 ℃ overnight, and the diameter of the bacteriostatic circle is recorded. The results are shown in Table 5.

As can be seen from Table 5, 2 strains of lactic acid bacteria fermentation broth had zones of inhibition on the plate. The lactobacillus plantarum KS1-1 can inhibit the growth of escherichia coli and staphylococcus aureus simultaneously, and the lactococcus lactis ZJL1-3 can inhibit the growth of escherichia coli, so that the two lactobacillus strains can be applied to fermentation of the fermented bean curd bittern to effectively improve the pollution of pathogenic bacteria in the fermentation process.

TABLE 5 determination of the zone of inhibition of the strains

Example 8 and 4-Strain lactic acid bacteria compounding and direct vat set fermentation of fermented Bean curd marinade

The method is characterized in that 4 strains of lactic acid bacteria are compounded and used for fermenting the stock solution of the brine, and the method comprises the following specific steps:

(1) freeze-dried powder of 4 strains of lactic acid bacteria is taken and compounded in equal amount to be used as a leavening agent.

(2) Preparing a brine stock solution, wherein the components of the brine stock solution comprise amaranth stems, ginger, pepper and sterile saline water, the concentration of the amaranth stems is 12 g/100mL, the concentration of the ginger is 1.25g/100mL, the concentration of the pepper is 0.63g/100mL, and the concentration of the sterile saline water is 2g/100 mL;

(3) inoculating a proper amount of the leavening agent obtained in the step (1) into the stock solution of the halogen solution obtained in the step (2); culturing the stock solution of the marinating solution at 37 ℃ for 12h, adding the ground bean curd blanks as a fermentation nitrogen source according to the volume ratio of 2%, and continuously fermenting at 37 ℃ for 12h to obtain the marinating solution of the stinky bean curd; the initial composite strain total bacterial concentration of the leavening agent in the stock solution of the halogen solution is about 10⁴CFU/mL。

In this example, lactobacillus plantarum KS1-1, lactobacillus plantarum KS1-2, pediococcus pentosaceus ZJL2-2, and lactobacillus lactis ZJL1-3 were mixed and fermented in equal amounts, and after 0h, 6h, 12h, 18h, and 24h, respectively, the stinky tofu halogen solution samples were plated to count colonies, and the growth curve, pH, and color change of the fermentation broth were measured. The results are shown in FIGS. 2 to 4.

As can be seen from FIG. 2, the 4 strains of lactic acid bacteria were subjected to complex fermentation, and then fermented for about 12-18 h, and then entered the stationary phase, and the bacterial concentration reached 10 at 18h of fermentation¹⁴CFU/mL. The bacterial concentration reaches 10 after 24 hours of fermentation^14.5CFU/mL. As can be seen from the comparison between fig. 2 and fig. 1, the fermentation capacity of the starter after the combination of 4 strains of lactic acid bacteria is significantly improved compared to that of lactic acid bacteria alone as a starter, and the bacterial concentration at the end of fermentation is significantly higher than that of single strain of lactic acid bacteria.

The acidity change of the fermented bean curd marinating liquid in the fermentation process is analyzed, as shown in fig. 3. The acid production capacity of the 4 lactic acid bacteria in the early stage of fermentation is strong, the pH of the fermentation liquor is gradually reduced, the acidity reaches the highest value in 12 hours, the pH of the fermentation liquor is reduced to 3.17 at the moment, and the titration acidity reaches 13.1 degree T/10 mL. The pH value gradually rises in the middle and later stages of fermentation, the pH value of the fermentation liquor reaches 5.32 when the fermentation liquor is 24 hours, and the titration acidity of the stinky tofu halogen liquor is recovered to 9.3 degrees T.

Because the excessive acidity tends to promote the deterioration of bean products and brings stimulation, strong and unpleasant sour and astringent feelings, in the process of putting the bean curd blanks into the final product of the fermented bean curd marinating liquid to ferment and produce the fermented bean curd products, the putting and soaking of the bean curd blanks are carried out for 6-12 h without the high acidity, and the putting can be carried out for 18-24 h, so that the unpleasant sensory influence on the fermented bean curd products is reduced.

At the same time, the color change of the fermentation broth during fermentation was observed and recorded as shown in FIG. 4. As can be seen from fig. 4, the original halogen solution stock solution is greenish and has high transparency. Gradually becomes yellow and brown in the fermentation process. After the ground bean curd blanks are added, the transparency is gradually reduced, the fermentation liquor becomes dark brown when the fermentation time is 18-20 h, the transparency is lower, and the color and the appearance of the fermented strong-smelling fermented bean curd are very close to those of naturally fermented commercial strong-smelling fermented bean curd marinating liquor. In conclusion, the optimal fermentation period of the halogen solution is selected to be 18-20 h at 37 ℃.

Examples 9-14, 4 Strain lactic acid bacteria compounding and direct vat set fermentation of fermented Bean curd marinade

The fermentation process of examples 9-14 was substantially the same as that of example 8, and the results are shown in Table 7 with the changed fermentation conditions shown in Table 6.

TABLE 6 fermentation Process parameters for examples 9-14

TABLE 7 fermentation product Properties of examples 9-14

Observing that the color change of the fermentation liquid in the fermentation processes of the examples 9-14 is basically the same as that in the example 8, the fermentation liquid becomes dark brown after 20 hours of fermentation, the transparency is lower, and the color and the appearance of the fermented brine of the strong-smelling preserved bean curd are very close to those of the naturally fermented commercial fermented brine of the strong-smelling preserved bean curd.

In summary, the properties of the fermented bean curd marinade obtained under the fermentation process conditions of examples 9-14 are all close to those of naturally fermented bean curd marinade. And 4 specific lactic acid bacteria are compounded in equal amount to serve as a leavening agent for fermentation, so that the fermentation of the fermented bean curd marinating liquid can be effectively promoted, and the fermentation time is shortened.

In the embodiment, the compound ratio ranges of lactobacillus plantarum KS1-1, lactobacillus plantarum KS1-2, pediococcus pentosaceus ZJL2-2 and lactococcus lactis ZJL1-3 are as follows: 1 (0.5-2), (1-3) and (0.5-4).

Example 15: analysis of volatile aroma components in strong-smelling fermented bean curd marinating liquid obtained by fermenting 4 single strains and performing compound fermentation on 4 lactobacillus strains

The volatile aroma components of the fermented bean curd halogen liquid products of the examples 3-6 and 8 are extracted by a Solid Phase Microextraction (SPME) method and analyzed and identified by GC-MS.

The 85 μm PDMS extraction head was aged at 250 deg.C for 12h before first use at GC-MS inlet, and then aged at 250 deg.C for 30min before use.

Gas chromatography detection conditions: performing gas chromatography analysis by using a DB-WAX chromatographic column, wherein the sample injection mode is non-shunting, the flow of carrier gas (He) is 1.0mL/min, and the temperature rise program is as follows: maintaining the initial temperature at 40 deg.C for 4 min; raising the temperature to 100 ℃ at 5 ℃/min, raising the temperature to 220 ℃ at 10 ℃/min, and keeping the temperature at 220 ℃ for 8 min.

Mass spectrum detection conditions: the ion source temperature is 200 ℃; an EI ionization source; the electron energy is 70 eV; the detector is 350V; the scanning range M/Z is 33 to 450 amu.

The specific detection steps are as follows: 30mL of single strain or mixed strain fermented strong-smelling preserved bean curd fermented brine product, 5g of sodium chloride and 5 mu L of phenethyl acetate with the concentration of 20 mu g/mL are put in a headspace extraction bottle, the height of a PDMS extraction head is adjusted to be 0.5cm above the liquid plane, the mixture is placed on a magnetic stirrer under the heating condition of 50 ℃, the stirring speed is set to be 80r/min, and extraction is carried out for 90 min. And after extraction is finished, quickly removing an extraction head, connecting the extraction head with a GC-MS sample inlet, desorbing the extraction head at the sample inlet for 5min at 250 ℃ for subsequent analysis, and detecting three parallel data of each sample.

The GC-MS analysis identified 16 main volatile aroma components in the single-strain fermented strong-smelling preserved bean curd marinade of examples 3-6, as shown in Table 8.

TABLE 8 main characteristic aroma components in single-strain fermented strong-smelling fermented bean curd bittern solution and relative content thereof

From the experimental results in table 8, it is clear that the odor components can be detected in the stinky tofu halogen liquid after single strain fermentation of lactobacillus plantarum KS1-1, lactobacillus plantarum KS1-2, pediococcus pentosaceus ZJL2-2 and lactobacillus lactis ZJL1-3, wherein the odor components have high contents of 4 types of alcohols, 4 types of esters, 2 types of aldehydes, 1 type of ketones and other 4 types. The lactobacillus plantarum KS1-1, lactobacillus plantarum KS1-2 and lactococcus lactis ZJL1-3 fermentation products are high in alcohol and ester volatile substances, the pediococcus pentosaceus ZJL2-2 fermentation products are high in indole and thioether substances, and the substances make a prominent contribution to aroma characteristic formation.

GC-MS analysis of the total ion flow graph in the mixed strain fermented strong-smelling preserved bean curd marinade of example 8 is shown in fig. 5 and 6, and 51 volatile aroma components are identified in total, as shown in Table 9.

TABLE 9 volatile aroma components of fermented bittern of strong-smelling fermented bean curd and relative content thereof

From the results in table 9, it was found that 61 kinds of volatile aroma components were identified in the fermented bean curd halogen solution obtained by the fermentation with lactic acid bacteria. Wherein, the composition comprises 19 kinds of olefins, 10 kinds of alcohols, 5 kinds of esters, 4 kinds of acids, 3 kinds of aldehydes, 3 kinds of ketones and 7 kinds of other compounds (including amines, thioethers, indoles, phenols and the like). The experiment is basically consistent with the analysis of the aroma components of the strong-smelling preserved bean curd marinating liquid in the prior art. The aroma component components were subjected to classification analysis as follows.

(1) Alkanes and hydrocarbons

The total number of alkane substances obtained by experimental identification is 19, wherein 13 alkenes, 1 hydrocarbon and 5 alkanes are contained. Most of alkene substances are aroma components which are characterized by aroma of fruit trees, rosin and oranges, which may come from fresh amaranth stems, spices such as ginger and pepper, and the abundance of the alkene substances in the raw material liquid before fermentation is high, and the abundance of the alkene substances in the fermented strong-smelling bean curd marinating liquid after fermentation is obviously reduced. The cis-alpha-bergamotene is obviously reduced to 2.41 percent from 22.5 percent before fermentation, and the beta-bisabolene is reduced to 1.68 percent from 7.32 percent. The aromatic threshold of the alkane compounds is high, the alkane compounds do not have flavor activity, the abundance after fermentation is generally reduced, and the aroma characteristics of the fermented bittern liquid of the strong-smelling preserved bean curd are slightly contributed.

(2) Alcohols

The carbohydrate is converted into alcohol by lactic acid bacteria during fermentation. It is worth noting that most of the alcohol substances are low in abundance before fermentation and are increased after fermentation. The changes are more obvious that n-butanol is changed from 0% to 38.3%, eucalyptol is changed from 2.39% to 5.42%, linalool is changed from 4.08% to 8.76%, and 4-terpene alcohol is changed from 6.51% to 7.98%. The n-butyl alcohol has mellow fragrance, the eucalyptus mellow fragrance is characterized by camphor fragrance, the linalool is characterized by muguet fragrance, and the 4-terpene alcohol fragrance is characterized by wood and soil fragrance, and the fragrances can endow the fermented strong-flavor bean curd marinade with mellow fragrance to form important fragrance characteristics of the strong-flavor bean curd marinade. The 4 specific lactic acid bacteria are subjected to compound fermentation, so that the abundance of most alcohol substances can be improved, particularly the abundance of n-butyl alcohol is greatly improved, and the fragrance of the stinky tofu marinating liquid is improved.

(3) Esters as pesticides

The experimental identification shows that the total number of the obtained esters is 5, the esters are hardly detected in the raw material of the stinky tofu halogen liquid before fermentation, and the esters except butyl acetate are all generated in the fermentation process. Among the esters contributing to aroma formation are butyl acetate (abundance 1.78%), hexyl acetate (abundance 0.74%), ethyl valerate (abundance 1.12%) and 3-octenyl ester (abundance 1.35%). The esters are mostly radix aucklandiae and sweet fragrance. Although the abundance of the ester compound is low, the aromatic threshold of the ester compound is very low, and the ester compound can still be considered as an important component for the characteristic aroma of the brine.

(4) Acids (acids)

The acid substances are mainly generated by the hydrolysis of soybean lipid and have sour taste and oil smell. The total number of the acid substances obtained by experimental identification is 4, the total abundance is low, most of the acid compounds are reduced in abundance or even disappear in the fermentation process, and only the abundance of butyric acid is increased, and the butyric acid is a short-chain fatty acid which is generated by lactic acid bacteria and is beneficial to health. Wherein 2-methyl valeric acid and isovaleric acid are generated by degrading amino acid with lactic acid bacteria, 2-methyl valeric acid has cheese smell, isovaleric acid has unpleasant rancidity smell, but the abundance of the two acid compounds is very low. The acid compound has little influence on the aroma characteristics of the brine and exists mainly as a background odor.

(5) Aldehydes and ketones

The aldehydes identified by the experiment are 3 in total, and are not detected in the raw material liquid before fermentation and are all generated in the fermentation process. Wherein the characteristic fragrance of citral (0.42%) and neral (0.21%) is lemon fruit fragrance, and can be generated by oxidation of alcohols or phenols. The ketone substances obtained by identification are 3 types, 3-octanone (1.78%) has fruit fragrance, cis-dihydroartemisinine (0.31%) is usually detected in blueberry fruits, 2-tridecanone has dairy and coconut fragrance (0.27%), and the ketone compounds have important influence on the fragrance characteristics of the brine.

(6) Others

The other types of substances obtained by experimental identification are 7 in total, have higher abundance in the fermented halogen solution, are also important components of the characteristic aroma of the halogen solution, and are mostly generated in the fermentation process. The pesticide comprises 2 amines, 3 thioethers, 1 phenol and indole. The amine substance has ammonia smell, and the thioether substance is obtained by degrading sulfur-containing amino acid by lactic acid bacteria. The dimethyl diether (6.32%), dimethyl trisulfide (1.40%) and dimethyl tetrasulfide (1.11%) identified in this example have low aroma thresholds, have onion and radish odors, are detectable in allium species, fermented soybeans and mustard, and sulfur-containing compounds essentially contribute to the aroma characteristics of foods. Indole (27.9%) is generated by degrading amino acid by microorganisms, has high abundance in halogen liquid, strong excrement odor, low aromatic threshold value as a heterocyclic substance and jasmine flower fragrance only under diluted low concentration (Ames JM. EUCOST action 919 melanoides in food and technology. Interactive compositions 2002,1245: 389-; congratulating et al reported that indole was not detected in the raw materials for the fermentation of the brine, but was always present in the fermentation process and was a specific component of the fermentation broth; liuyuping et al (Liuyuping, Chenhaitao, Sun Bao, et al. solid phase micro extraction and GC-MS method analysis of volatile components [ J ] in fermented bean curd for Food industry science, 2009, 30 (12): 403 + 405.), Sun Jie Wen et al (Sun Ji Wen, Yankee, Liyanmin. SDE in combination with GC-MS analysis of characteristic aroma components [ J ] in Wang and stinky tofu for Food science 2015,36(16):127 + 131.) and Zhengxiaofen et al (SHIIU H C, YASUAKI T, KOICHI W, et al.

The analysis result shows that the main aroma components of the stinky tofu halogen liquid in the invention are 2-carene, gamma-terpinene, cis-alpha-bergamotene, n-butyl alcohol, eucalyptol, linalool, 4-terpene alcohol, alpha-terpineol, butyl acetate, hexyl acetate, ethyl valerate, 3-octenyl ester, 2-methylvaleric acid, citral, neral, 3-octanone, cis-dihydroxanthene ketone and 2-tridecanone. Typical aroma components of the stinky tofu marinating liquid are dimethyl diether, dimethyl trisulfide, dimethyl tetrasulfide and indole. The main components of the volatile aroma components of the stinky tofu halogen liquid are very similar to the typical reported typical commercially available naturally fermented stinky tofu halogen liquid, which shows that the aroma characteristics of the stinky tofu halogen liquid product are closer to those of the traditional product, and the basic requirements of consumers on the aroma characteristics can be better met.

The volatile aroma components of the fermented bean curd halogen liquid products of the examples 9 to 14 are extracted by the same method, and the analysis and identification are carried out by GC-MS, and the result is similar to the analysis result of the volatile aroma components of the example 8.

In conclusion, the aroma components of the fermented bean curd marinated liquid product obtained by equally compounding 4 lactic acid bacteria serving as a leavening agent are obviously more than those of a single-strain fermented product, and the abundance of the characteristic aroma components is higher. The fermentation raw material generates or converts aroma components in the metabolic process of the compound lactobacillus, and the formation of the aroma characteristics of the fermented bean curd marinating liquid is closely related to the compounding of the lactobacillus.

Example 16 soaking of Strong-smelling fermented Bean curd

The strong-smelling fermented bean curd A is obtained by soaking bean curd in the fermented bean curd marinade of example 8 for 12 hours. And simultaneously soaking the same amount of bean curd in the same volume of commercially available natural fermentation type stinky tofu marinating solution for 24 hours to obtain stinky tofu B. The results of evaluation of fermented bean curd a and fermented bean curd B by 50 volunteers are shown in table 10.

TABLE 10 sensory test evaluation results

The results in table 10 show that the odor and the quality of the strong-smelling preserved bean curd of the invention are similar to or even better than those of the strong-smelling preserved bean curd soaked in the natural fermentation type strong-smelling preserved bean curd marinade in the market.

In conclusion, the fermented bean curd marinating liquid prepared by equivalently compounding 4 specific lactic acid bacteria serving as a leavening agent through fermentation has the advantages of short fermentation period, fixed formula, definite fermentation strain, stable aroma quality and the like, and is suitable for being popularized in industrial production. The aroma of the strong-smelling fermented bean curd is similar to that of the commercial fermented bean curd marinating liquid, the traditional requirement of the market on the flavor can be met, and the strong-smelling fermented bean curd marinating liquid has a wide market application value.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims

1. A starter culture is characterized in that the starter culture is compounded by 4 strains of lactic acid bacteria; wherein:

2. The starter culture according to claim 1, wherein the starter culture is prepared by mixing 4 strains of lactic acid bacteria in equal amount.

3. The starter culture according to claim 1, wherein the lactic acid bacteria is freeze-dried powder of lactic acid bacteria.

4. The leavening agent according to any one of claims 1 to 3, wherein the leavening agent is used for fermenting a marinade stock solution to prepare a marinade of strong-smelling preserved bean curd.

5. The direct-vat-set fermentation method of the stinky tofu marinating liquid is characterized by comprising the following steps:

inoculating the starter culture of any of examples 1-4 into a halogen liquor stock solution, the starter culture being initially in the halogen liquor stock solutionTotal concentration of 10^3-6CFU/mL, culturing at 30-45 ℃ for 10-15 h, then adding a fermentation nitrogen source according to the volume ratio of 1-5%, and continuing to ferment for 10-15 h to obtain the stinky tofu marinating liquid.

6. The direct vat set fermentation method of strong-smelling preserved bean curd according to claim 5, wherein the 4 strains of lactic acid bacteria are equivalently compounded in the form of freeze-dried powder of lactic acid bacteria as the leavening agent.

7. The direct vat set fermentation method of strong-smelling preserved bean curd according to claim 5, wherein the components of the stock solution of the strong-smelling preserved bean curd include amaranth stems, ginger, pepper and sterile saline; in the brine stock solution, the concentration of the amaranth stalks is 10-50 g/100mL, the concentration of the ginger is 1-10 g/100mL, the concentration of the pepper is 0.5-5 g/100mL, and the concentration of the sterile saline water is 1-5 g/100 mL.

8. The direct vat set fermentation method of strong-smelling preserved bean curd according to claim 7, wherein the concentration of the amaranth stem is 12.25g/100mL, the concentration of the ginger is 1.25g/100mL, and the concentration of the pepper is 0.63g/100mL in the stock solution of the strong-smelling preserved bean curd; the concentration of the sterile saline is 2g/100 mL; the initial total concentration of the leavening agent in the stock solution of the brine is 10⁴CFU/mL。

9. The direct vat set fermentation method of strong-smelling preserved bean curd according to claim 5, wherein the stock solution of the strong-smelling preserved bean curd is cultured at 37 ℃ for 12 hours, and then a fermentation nitrogen source is added at a volume ratio of 2% to continue the fermentation for 12 hours to obtain the strong-smelling preserved bean curd.

10. A marinated strong-smelling preserved bean curd obtained by the direct vat fermentation method of a marinated strong-smelling preserved bean curd according to any one of claims 5 to 9.