CN111685305B - Method for producing soy sauce by fermentation of high-salt-tolerance lactobacillus - Google Patents
Method for producing soy sauce by fermentation of high-salt-tolerance lactobacillus Download PDFInfo
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
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- OTPDWCMLUKMQNO-UHFFFAOYSA-N 1,2,3,4-tetrahydropyrimidine Chemical compound C1NCC=CN1 OTPDWCMLUKMQNO-UHFFFAOYSA-N 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
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- GHFGJTVYMNRGBY-UHFFFAOYSA-N m-tyramine Chemical compound NCCC1=CC=CC(O)=C1 GHFGJTVYMNRGBY-UHFFFAOYSA-N 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/50—Soya sauce
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/27—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption
- A23L5/273—Removal of unwanted matter, e.g. deodorisation or detoxification by chemical treatment, by adsorption or by absorption using adsorption or absorption agents, resins, synthetic polymers, or ion exchangers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D36/00—Filter circuits or combinations of filters with other separating devices
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Polymers & Plastics (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Microbiology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Soy Sauces And Products Related Thereto (AREA)
Abstract
The technical scheme of the invention discloses a method for producing soy sauce by fermenting high-salt-tolerance lactobacillus, which comprises the steps of preparing a mixed culture medium, pre-culturing, collecting lactobacillus thalli, collecting salt-tolerance response reaction substances, preparing starter, preparing soy sauce mash, fermenting and squeezing and sterilizing; the lactobacillus is pre-cultured by a high-salt culture medium, and salt-tolerance response reaction substances are collected by ultrasonic crushing and used for soy sauce fermentation, so that the salt tolerance of the lactobacillus is obviously improved, the fermentation time of the soy sauce is shortened, and the flavor and quality of the soy sauce are improved.
Description
Technical Field
The invention relates to the technical field of food fermentation engineering, in particular to a method for producing soy sauce by fermenting high-salt-tolerance lactobacillus.
Background
Soy sauce is a traditional fermented seasoning in China, and is a seasoning with unique flavor, which is formed by taking soybeans, wheat, bran and the like as raw materials and fermenting for a long time under the combined action of a plurality of microorganisms. Soy sauce is one of the indispensable condiments in daily life, and the requirements of people on the taste and aroma of soy sauce are also increasing.
Lactic acid bacteria have been used in the fermentation of soy sauce, and different lactic acid bacteria decompose different amino acids in soy sauce, and representative lactic acid bacteria in soy sauce are Pediococcus halophilus, micrococcus sojae, lactobacillus plantarum, etc. The lactic acid bacteria have weak lactic acid resistance, so that the quality of the fermented grains is not reduced due to the fact that the pH of the fermented grains is too low due to the fact that excessive lactic acid is produced. However, the salt concentration is high in the soy sauce brewing process, and the high salt concentration is a main environmental stress in the lactobacillus fermentation process. The salt tolerance of different lactic acid bacteria is different, and even lactic acid bacteria with higher salt tolerance cannot avoid the problem of reduction of viable bacteria number along with fermentation in the soy sauce fermentation process.
An increase in the salt concentration of the solution results in an increase in the osmotic pressure of the solution, and the cells, to maintain normal turgor pressure, tend to initiate their own stress-resistant protective substances, known as compatible solutes. The compatible substance is an important stress-resistant protective substance in halophiles, however, in practical production, the application of adding compatible solutes to effectively improve the salt tolerance of lactic acid bacteria in the fermentation process is not available, so as to prevent the toxicity of the fermentation product from being increased. Especially, the soy sauce is taken in a large amount and continuously for a long time in daily life, so that the added substances are cautious in the soy sauce fermentation process, and adverse effects on human health are avoided. Patent CN 105361062B discloses a method for improving salt tolerance of lactobacillus, which is to add tetrahydropyrimidine, methionine, histidine, lysine or proline into fermented product to ferment, so that the viable count of lactobacillus can be improved by about 1 time under the sublethal concentration of salt, but excessive intake of amino acid and pyrimidine added into soy sauce may cause a certain toxicity to human body, or influence the immunity of human body, and the safety still has a certain problem. The existing method for screening the high-salt-tolerance lactobacillus strain has the disadvantages of large workload and high cost. Therefore, a safer method for improving the salt tolerance of lactobacillus is needed to be found, so that the lactobacillus strain can survive in the soy sauce brewing process, namely, the method can adapt to the soy sauce brewing process and can survive in the environment with high salt content, thereby meeting the use requirements of different soy sauce brewing processes.
Disclosure of Invention
In order to solve the prior art problems, the invention provides a method for producing soy sauce by fermenting high salt tolerance lactobacillus, which is characterized in that lactobacillus compatible solutes are prepared and added into the soy sauce fermentation process, so that the salt tolerance of the lactobacillus is improved, the fermentation time of the soy sauce is shortened, and the quality and flavor of the soy sauce are improved.
The technical scheme of the invention is as follows: a method for producing soy sauce by fermenting high-salt-tolerance lactobacillus comprises the following steps:
s1, preparing a mixed culture medium, namely preparing an MRS liquid culture medium with a salt content of 10%, adding a compatible solute composition into the culture medium, and uniformly mixing to obtain the mixed culture medium, wherein the concentration of the compatible solute composition in the mixed culture medium is 6-10 g/L;
the compatible solute composition includes an amino acid composition including at least two of proline, alanine, aspartic acid, threonine, and tryptophan, and an enzyme composition including at least one of glutamine transaminase, lactate dehydrogenase, and phosphofructokinase; the mass ratio of the amino acid composition to the enzyme composition in the compatible solute composition is (1.5-3) 1; in the amino acid composition, the proline is as follows by weight ratio: alanine: aspartic acid: threonine: tryptophan is 1 (0.3-0.8): 0-0.5): 0-0.3; in the enzyme composition, the weight ratio of glutamine transaminase to lactic dehydrogenase to phosphofructokinase is 1 (0-0.5): 0-0.3);
s2, pre-culturing, namely inoculating lactobacillus cultured to a logarithmic phase into a mixed culture medium, and culturing at the constant temperature of 30-35 ℃ for 24-72 hours;
the lactic acid bacteria comprise pediococcus halophilus, micrococcus sojae, lactococcus lactis, pediococcus acidilactici and lactobacillus plantarum, and the weight ratio of the pediococcus halophilus is as follows: micrococcus soyabean sauce: lactococcus lactis: pediococcus acidilactici: the lactobacillus plantarum is 1 (0.5-1) (0.3-0.5) (0.2-0.5) (0.3-0.8);
s3, collecting lactobacillus thallus, centrifugally collecting the pre-cultured lactobacillus to obtain the lactobacillus thallus, flushing 3 times by using 0.075M PBS buffer solution, and discarding the supernatant;
the rotation speed of centrifugal collection is 6000rpm, the time is 10-20 min, and the temperature is 4-10 ℃; the mass ratio of the volume of the PBS buffer solution to the lactobacillus thallus is (50-80) ml to 1g;
s4, collecting salt-resistant response reaction substances, performing ultrasonic crushing on the washed lactobacillus cells, centrifuging, collecting supernatant after ultrasonic crushing, and performing membrane filtration sterilization to obtain salt-resistant response reaction substances;
the power of ultrasonic crushing is 300-350W, the ultrasonic is performed for 5s, the ultrasonic is stopped for 5s, and the ultrasonic time is 30-50 min; collecting supernatant after ultrasonic crushing, wherein the centrifugal speed is 12000-15000 rpm, the time is 10-15 min, and the temperature is 4-10 ℃; the pore diameter of the membrane filtration is 0.22 mu m;
s5, starter propagation, namely, stir-frying the wheat, crushing the wheat to prepare wheat flour, and moistening the soybean meal; continuously steaming the soybean meal after wetting water, wherein the steaming temperature is 130 ℃, and the steaming time is 5-15 min; mixing steamed bean pulp and wheat flour according to the weight ratio of (1.2-1.6) to 1, adding lactic acid, adjusting the pH of the mixture of the bean pulp and the wheat flour to be 5.5-6.0, inoculating Aspergillus oryzae, and fermenting in a pool to obtain fermented grain koji;
s6, preparing soy sauce mash, namely mixing the prepared fermented grain yeast with salt water with the concentration of 20-25 g/100ml, and then pumping the mixture into a fermentation tank to prepare the soy sauce mash, wherein the weight ratio of the fermented grain yeast to the salt water is 1 (1.5-1.8);
s7, fermenting, namely adding the salt tolerance response reaction substance prepared in the step S4, the lactobacillus fermentation strain and yeast into a fermentation tank, fermenting at 15-20 ℃ for 10 days, heating to 27-30 ℃, fermenting for 60 days, cooling to 20-25 ℃ and fermenting for 10 days to obtain the mature soy sauce mash;
the mass ratio of the salt tolerance response reaction substance to the lactobacillus fermentation strain to the yeast to the soy sauce mash is 1 (28-35): 300-500): 1000-12000; the lactobacillus fermentation strain comprises at least one of Pediococcus halophilus, micrococcus sojae, lactococcus lactis, pediococcus acidilactici and Lactobacillus plantarum; the yeast is mixed yeast powder of the roux yeast and the globus hystericus, and the roux yeast is prepared from the following materials in percentage by weight: the globoid yeast is (5-6) 1-2;
s8, squeezing and sterilizing, namely squeezing the mature soy sauce mash according to a squeezing process to obtain raw soy sauce, filtering the raw soy sauce by diatomite and microporous membranes, sterilizing, transferring the sterilized raw soy sauce into a precipitation tank, standing for storage, precipitating for 30 days, and sub-packaging;
the fineness of the diatomite is 10# and 95 ℃; the particle size of the microporous membrane is 0.45 mu m; the sterilization temperature is 95 ℃ and the sterilization time is 20-30 min.
When the salt content in the culture medium is higher, the extracellular osmotic pressure is increased, and the lactic acid bacteria maintain the normal osmotic pressure of cells by accumulating compatible solute compositions, change glycolytic metabolic pathways and the like, regulate intracellular metabolic balance and maintain the normal growth of the lactic acid bacteria so as to realize the improvement of the salt tolerance of the lactic acid bacteria. The invention adopts the high-salt culture medium to pre-culture the lactobacillus, so that the lactobacillus induces a series of stress response reactions, activates various enzymes and related substances participating in related metabolic pathways of the lactobacillus, collects the substances which are activated and related to salt tolerance, namely salt tolerance response reaction substances in the lactobacillus, and is used in the soy sauce fermentation process, thereby obviously improving the salt tolerance of the lactobacillus, extracting the substances from the lactobacillus, being safe, nontoxic and harmless, and preventing the harmful effects on human bodies caused by excessive content of substances such as amino acid and the like additionally added in the soy sauce. The invention increases the salt tolerance of lactobacillus by the compatible solute composition, so that the fermentation time of the soy sauce is obviously shortened, and the flavor and the quality are better.
By adopting the technical scheme, the beneficial effects of the invention are as follows:
(1) The invention pre-cultures lactobacillus by a high-salt culture medium, ultrasonically breaks and collects salt-tolerance response reaction substances, is used in the soy sauce fermentation process, has high food safety, avoids adverse effects on human bodies caused by excessive additives, obviously enhances the salt tolerance of the lactobacillus, obviously shortens the soy sauce fermentation time, and improves the flavor and quality of the soy sauce.
(2) The raw materials are easy to obtain, the preparation method is simple, and the production and the application are facilitated.
(3) The high salt tolerance of the lactic acid bacteria can be realized without screening new strains, the time consumption is short, a large amount of manpower and material resources are saved, and the cost is low.
(4) The raw soy sauce is filtered by the diatomite and the microporous membrane and then sterilized and deposited to obtain a soy sauce finished product, and the diatomite and the microporous membrane are filtered to remove part of impurities in the raw soy sauce, and part of salt tolerance response reaction substances are filtered and adsorbed to further reduce the additive substances in the final soy sauce finished product, so that the soy sauce is safer for human bodies.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1
A method for producing soy sauce by fermenting high-salt-tolerance lactobacillus comprises the following steps:
s1, preparing a mixed culture medium, namely preparing an MRS liquid culture medium with a salt content of 10%, adding a compatible solute composition into the culture medium, and uniformly mixing to obtain the mixed culture medium, wherein the concentration of the compatible solute composition in the mixed culture medium is 8g/L;
the compatible solute composition comprises an amino acid composition and an enzyme composition, and the mass ratio of the amino acid composition to the enzyme composition in the compatible solute composition is 2:1; the amino acid composition comprises proline, alanine and aspartic acid in a mass ratio of 1:0.5:0.3; the enzyme composition comprises glutamine transaminase and lactic dehydrogenase in a mass ratio of 1:0.2;
s2, pre-culturing, namely inoculating lactobacillus cultured to a logarithmic phase into a mixed culture medium, and culturing at the constant temperature of 33 ℃ for 36 hours;
the lactic acid bacteria comprise pediococcus halophilus, micrococcus sojae, lactococcus lactis, pediococcus acidilactici and lactobacillus plantarum, and the weight ratio of the pediococcus halophilus is as follows: micrococcus soyabean sauce: lactococcus lactis: pediococcus acidilactici: lactobacillus plantarum is 1:0.8:0.4:0.3:0.5;
s3, collecting lactobacillus thallus, centrifugally collecting the pre-cultured lactobacillus to obtain the lactobacillus thallus, flushing 3 times by using 0.075M PBS buffer solution, and discarding the supernatant;
the rotation speed of centrifugal collection is 6000rpm, the time is 15min, and the temperature is 6 ℃; the mass ratio of the volume of the PBS buffer solution to the lactobacillus thallus is 70 ml/1 g;
s4, collecting salt-resistant response reaction substances, performing ultrasonic crushing on the washed lactobacillus cells, centrifuging, collecting supernatant after ultrasonic crushing, and performing membrane filtration sterilization to obtain salt-resistant response reaction substances;
the power of ultrasonic crushing is 330W, ultrasonic treatment is carried out for 5s, stopping for 5s, and the ultrasonic treatment time is 40min; collecting supernatant after ultrasonic crushing, wherein the centrifugal speed is 13000rpm, the time is 13min, and the temperature is 6 ℃; the pore diameter of the membrane filtration is 0.22 mu m;
s5, starter propagation, namely, stir-frying the wheat, crushing the wheat to prepare wheat flour, and moistening the soybean meal; continuously steaming the soybean meal after wetting water, wherein the steaming temperature is 130 ℃, and the steaming time is 10min; mixing steamed bean pulp and wheat flour according to a weight ratio of 1.4:1, adding lactic acid, adjusting the pH of the mixture of the bean pulp and the wheat flour to 5.8, inoculating Aspergillus oryzae, and fermenting in a pool to obtain fermented grain koji;
s6, preparing soy sauce mash, namely mixing the prepared fermented grain yeast with brine with the concentration of 23g/100ml, and then pumping the mixture into a fermentation tank to prepare the soy sauce mash, wherein the weight ratio of the fermented grain yeast to the brine is 1:1.6;
s7, fermenting, namely adding the salt tolerance response reaction substance prepared in the step S4, the lactobacillus fermentation strain and yeast into a fermentation tank, fermenting at the temperature of 18 ℃ for 10 days, heating to 28 ℃, fermenting for 60 days, cooling to 23 ℃ and fermenting for 10 days to obtain mature soy sauce mash;
the mass ratio of the salt tolerance response reaction substance to the lactobacillus fermentation strain to the yeast to the soy sauce mash is 1:30:400:11000; the lactobacillus fermentation strain comprises micrococcus sojae and lactobacillus plantarum, and the mass ratio is 1:2; the yeast is mixed yeast powder of the roux yeast and the globus hystericus, and the roux yeast is prepared from the following materials in percentage by weight: the ratio of the torulopsis yeast to the torulopsis yeast is 5:2;
s8, squeezing and sterilizing, namely squeezing the mature soy sauce mash according to a squeezing process to obtain raw soy sauce, filtering the raw soy sauce by diatomite and microporous membranes, sterilizing, transferring the sterilized raw soy sauce into a precipitation tank, standing for storage, precipitating for 30 days, and sub-packaging;
the fineness of the diatomite is 10# and 95 ℃; the particle size of the microporous membrane is 0.45 mu m; the sterilization temperature is 95 ℃ and the sterilization time is 25min.
Example 2
A method for producing soy sauce by fermenting high-salt-tolerance lactobacillus comprises the following steps:
s1, preparing a mixed culture medium, namely preparing an MRS liquid culture medium with a salt content of 10%, adding a compatible solute composition into the culture medium, and uniformly mixing to obtain the mixed culture medium, wherein the concentration of the compatible solute composition in the mixed culture medium is 10g/L;
the compatible solute composition comprises an amino acid composition and an enzyme composition, and the mass ratio of the amino acid composition to the enzyme composition in the compatible solute composition is 3:1; the amino acid composition comprises proline and alanine in a mass ratio of 1:0.8; the enzyme composition is glutamine transaminase;
s2, pre-culturing, namely inoculating lactobacillus cultured to a logarithmic phase into a mixed culture medium, and culturing at the constant temperature of 30 ℃ for 72 hours;
the lactic acid bacteria comprise pediococcus halophilus, micrococcus sojae, lactococcus lactis, pediococcus acidilactici and lactobacillus plantarum, and the weight ratio of the pediococcus halophilus is as follows: micrococcus soyabean sauce: lactococcus lactis: pediococcus acidilactici: lactobacillus plantarum is 1:0.5:0.3:0.5:0.3;
s3, collecting lactobacillus thallus, centrifugally collecting the pre-cultured lactobacillus to obtain the lactobacillus thallus, flushing 3 times by using 0.075M PBS buffer solution, and discarding the supernatant;
the rotation speed of centrifugal collection is 6000rpm, the time is 20min, and the temperature is 4 ℃; the mass ratio of the volume of the PBS buffer solution to the lactobacillus thallus is 50ml to 1g;
s4, collecting salt-resistant response reaction substances, performing ultrasonic crushing on the washed lactobacillus cells, centrifuging, collecting supernatant after ultrasonic crushing, and performing membrane filtration sterilization to obtain salt-resistant response reaction substances;
the power of ultrasonic crushing is 300W, ultrasonic treatment is carried out for 5s, stopping for 5s, and the ultrasonic treatment time is 30min; collecting supernatant after ultrasonic crushing, wherein the centrifugal speed is 12000rpm, the time is 10min, and the temperature is 4 ℃; the pore diameter of the membrane filtration is 0.22 mu m;
s5, starter propagation, namely, stir-frying the wheat, crushing the wheat to prepare wheat flour, and moistening the soybean meal; continuously steaming the soybean meal after wetting water, wherein the steaming temperature is 130 ℃ and the steaming time is 15min; mixing steamed bean pulp and wheat flour according to a weight ratio of 1.2:1, adding lactic acid, adjusting the pH of the mixture of the bean pulp and the wheat flour to 6.0, inoculating Aspergillus oryzae, and fermenting in a pool to obtain fermented grain koji;
s6, preparing soy sauce mash, namely mixing the prepared fermented grain yeast with 25g/100ml of salt water, and then pumping the mixture into a fermentation tank to prepare the soy sauce mash, wherein the weight ratio of the fermented grain yeast to the salt water is 1:1.5;
s7, fermenting, namely adding the salt tolerance response reaction substance prepared in the step S4, the lactobacillus fermentation strain and the yeast into a fermentation tank, fermenting at 15 ℃ for 10 days, heating to 30 ℃, fermenting for 60 days, cooling to 25 ℃ and fermenting for 10 days to obtain mature soy sauce mash;
the mass ratio of the salt tolerance response reaction substance to the lactobacillus fermentation strain to the yeast to the soy sauce mash is 1:35:300:1000; the lactobacillus fermentation strain is Pediococcus halophilus; the yeast is mixed yeast powder of the roux yeast and the globus hystericus, and the roux yeast is prepared from the following materials in percentage by weight: the ratio of the torulopsis yeast to the torulopsis yeast is 6:1;
s8, squeezing and sterilizing, namely squeezing the mature soy sauce mash according to a squeezing process to obtain raw soy sauce, filtering the raw soy sauce by diatomite and microporous membranes, sterilizing, transferring the sterilized raw soy sauce into a precipitation tank, standing for storage, precipitating for 30 days, and sub-packaging;
the fineness of the diatomite is 10# and 95 ℃; the particle size of the microporous membrane is 0.45 mu m; the sterilization temperature is 95 ℃ and the sterilization time is 30min.
Comparative example 1
Comparative example 1 differs from example 1 in that S1-S4 were deleted, starting from S5, S7 was fermentation, adding the compatible solute composition, lactic acid bacteria fermentation strain and yeast to a fermenter, fermenting at 15 ℃ for 10 days, then heating to 30 ℃, fermenting for 60 days, and then cooling to 25 ℃ for 10 days to obtain a mature moromi sauce;
the mass ratio of the compatible solute composition to the lactobacillus fermentation strain to the yeast to the soy sauce mash is 1:35:300:1000; the lactobacillus fermentation strain is Pediococcus halophilus; the yeast is mixed yeast powder of the roux yeast and the globus hystericus, and the roux yeast is prepared from the following materials in percentage by weight: the ratio of the torulopsis yeast to the torulopsis yeast is 6:1;
the compatible solute composition comprises an amino acid composition and an enzyme composition, and the mass ratio of the amino acid composition to the enzyme composition in the compatible solute composition is 2:1; the amino acid composition comprises proline, alanine and aspartic acid in a mass ratio of 1:0.5:0.3; the enzyme composition comprises glutamine transaminase and lactic dehydrogenase in a mass ratio of 1:0.2.
Blank control
The blank differs from example 1 in that S1-S4 are deleted, starting from S5, and no salt tolerance response material is added in S7.
1. Salt tolerance analysis test of lactic acid bacteria
Taking 0.5ml of each sample liquid from the fermented samples in step S7 in the examples 1, 2, 1 and the blank control, sampling, wherein the sampling time is respectively 10 days of fermentation, 70 days of fermentation and 80 days of fermentation, adding 4.5ml of labor-saving saline after sampling, and sequentially carrying out gradient dilution with the dilution factor of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 Plating with a suitable dilution gradient, eachThe gradients were repeated three times and the number of viable bacteria was counted, and the results are shown in Table 1.
TABLE 1 viable count of lactic acid bacteria during fermentation of soy sauce (cfu/cm) 3 )。
| Group of | Day 10 of fermentation | Day 70 of fermentation | Day 80 of fermentation |
| Example 1 | 3.4×10 3 | 7.3×10 8 | 9.6×10 9 |
| Example 2 | 3.1×10 3 | 6.9×10 8 | 8.1×10 9 |
| Comparative example 1 | 2.1×10 3 | 1.4×10 7 | 6.2×10 7 |
| Blank control | 1.3×10 2 | 3.7×10 6 | 5.8×10 6 |
As can be seen from the results in Table 1, the blank and comparative example 1 showed a small number of viable bacteria at 10, 70 and 80 days of fermentation, while the examples 1 and 2 showed a large number of viable bacteria and a high rate of increase in the number, indicating that the examples 1 and 2 had a high rate of proliferation and a large number of viable bacteria during fermentation of soy sauce, and further indicating that the examples 1 and 2 showed a high salt tolerance of lactic acid bacteria.
2. Test of the Effect of lactic acid bacteria on soy fermentation time
The total acid, amino nitrogen and reducing sugar contents were measured during the fermentation of soy sauce, and the results are shown in tables 2 to 4. Wherein the fermentation conditions are the same from 71 to 120 days of fermentation.
TABLE 2 Total acid content during fermentation of Soy sauce (g/100 ml)
TABLE 3 Ammonia-state nitrogen content during soy sauce fermentation (g/100 ml)
| Group of | Day 10 | Day 30 | Day 50 | Day 70 | Day 80 | Day 100 | Day 120 |
| Example 1 | 0.61 | 0.79 | 0.94 | 1.01 | 1.10 | 1.14 | 1.15 |
| Example 2 | 0.60 | 0.78 | 0.91 | 0.99 | 1.06 | 1.10 | 1.10 |
| Comparative example 1 | 0.58 | 0.69 | 0.84 | 0.86 | 0.88 | 0.89 | 0.90 |
| Blank control | 0.51 | 0.64 | 0.76 | 0.77 | 0.74 | 0.79 | 0.79 |
TABLE 4 reducing sugar content during fermentation of Soy sauce (g/100 ml)
| Group of | Day 10 | Day 30 | Day 50 | Day 70 | Day 80 | Day 100 | Day 120 |
| Example 1 | 7.2 | 5.9 | 4.6 | 3.1 | 2.2 | 2.1 | 2.1 |
| Example 2 | 7.3 | 5.8 | 4.4 | 3.2 | 2.4 | 2.3 | 2.2 |
| Comparative example 1 | 7.7 | 5.9 | 4.8 | 3.4 | 3.1 | 2.9 | 2.8 |
| Blank control | 7.9 | 6.1 | 5.0 | 4.2 | 4.1 | 3.9 | 3.9 |
As can be seen from the results of table 2, the total acid content and the amino nitrogen content of examples 1 and 2 gradually increased during the fermentation of soy sauce, the reducing sugar content gradually decreased, and the soy sauce was stable on day 80; the comparative example and the blank were gradually smoothed on days 50-70, and the total acid content and amino nitrogen content of examples 1 and 2 were higher, and the reducing sugar content was lower, probably because lactic acid bacteria were more rapidly proliferated and fermented, and lactic acid bacteria were more active, more abundant, and better in fermentation effect, so that lactic acid was produced, the amino nitrogen content was more produced, the amount of the metabolizing reducing sugar was more, the total acid amount and amino nitrogen amount were higher, and the growth rate was fast, and the reducing sugar amount was less, and the reduction rate was fast, in examples 1 and 2. The total acid content of the embodiment 1 and the embodiment 2 is below 1.1g/100ml, the amino nitrogen content reaches about 1.10g/100ml at 80 days, the requirements of national standards on the total acid content are met, and the amino nitrogen content is obviously higher than the first-level soy sauce standard, which indicates that the soy sauce quality is better.
When the present company ferments by the method of the blank group, the fermentation time is usually increased, and fermentation is usually selected for 100 days in order to make the fermentation effect better and the quality of soy sauce higher. In addition, as can be seen from the results of tables 2 to 4, the contents of total acid, amino nitrogen and reducing sugar were more stable at about 100 days of fermentation in the blank group, the values during the fermentation were relatively better, and the change in the contents was small as the fermentation time was prolonged. Therefore, the method of the present invention produces soy sauce, and by increasing the salt tolerance of lactic acid bacteria and applying it to the soy sauce fermentation process, the fermentation time of soy sauce can be reduced.
In addition, the amino nitrogen content of the soy sauce fermented for 80 days in the embodiment 1 and the embodiment 2 is higher, the delicate flavor of the soy sauce is improved, and the content of the reducing sugar is kept between 2 and 2.5g/100ml, so that the soy sauce has a certain sweet taste, and the whole taste of the soy sauce is fresh and sweet.
3. Assay of histamine and tyramine content in fermented soy sauce
The supernatant of the mature moromi on day 80 of the fermentation of examples 1, 2, comparative example 1 and blank was measured for the content of histamine and tyramine, and the results are shown in Table 5.
TABLE 5 Histamine and tyramine content (mg/L) in mature soy sauce mash
| Group of | Example 1 | Example 2 | Comparative example 1 | Blank control |
| Histamine | 16.7 | 18.2 | 24.1 | 29.3 |
| Tyramine | 7.6 | 8.1 | 22.4 | 30.7 |
As is clear from Table 5, the contents of histamine and tyramine in the soy sauce matured moromi of examples 1 and 2 are lower than those of comparative examples 1 and blank, and it is demonstrated that the methods for improving salt tolerance of lactic acid bacteria and fermenting soy sauce in examples 1 and 2 are safe, do not increase the amounts of two harmful substances of histamine and tyramine, can be used for soy sauce fermentation production, and have high product quality.
4. Sensory evaluation of Soy sauce
20ml of soy sauce produced in example 1, example 2, comparative example 1 and blank control were placed in 50ml of a clean beaker, and then evaluated by 20 professional evaluator in terms of color, aroma and taste, and the specific results are shown in Table 6.
TABLE 6 sensory evaluation effect of Soy sauce
As is clear from the results in Table 6, the soy sauce produced in example 1 and example 2 was more sweet and delicious than the soy sauce of comparative example 1 and the blank group, and the color was redder, brighter and glossy, indicating that example 1 and example 2 could improve the flavor and quality of the soy sauce.
Claims (8)
1. A method for producing soy sauce by fermenting high-salt-tolerance lactobacillus, which is characterized by comprising the following steps:
s1, preparing a mixed culture medium, namely preparing an MRS liquid culture medium with a salt content of 10%, adding a compatible solute composition into the culture medium, and uniformly mixing to obtain the mixed culture medium, wherein the concentration of the compatible solute composition in the mixed culture medium is 6-10 g/L, the compatible solute composition comprises an amino acid composition and an enzyme composition, the mass ratio of the amino acid composition to the enzyme composition is (1.5-3): 1, and in the enzyme composition, the mass ratio of glutamine transaminase to lactic dehydrogenase to phosphofructokinase is (0-0.5): 0-0.3) is 1;
s2, pre-culturing, namely inoculating lactobacillus cultured to a logarithmic phase into a mixed culture medium, and culturing at the constant temperature of 30-35 ℃ for 24-72 hours;
s3, collecting lactobacillus thallus, centrifugally collecting the pre-cultured lactobacillus to obtain the lactobacillus thallus, flushing 3 times by using 0.075M PBS buffer solution, and discarding the supernatant;
s4, collecting salt-resistant response reaction substances, performing ultrasonic crushing on the washed lactobacillus cells, centrifuging, collecting supernatant after ultrasonic crushing, and performing membrane filtration sterilization to obtain salt-resistant response reaction substances;
s5, starter propagation, namely, stir-frying the wheat, crushing the wheat to prepare wheat flour, and moistening the soybean meal; continuously steaming the soybean meal after wetting water, wherein the steaming temperature is 130 ℃, and the steaming time is 5-15 min; mixing steamed bean pulp and wheat flour according to the weight ratio of (1.2-1.6) to 1, adding lactic acid, adjusting the pH of the mixture of the bean pulp and the wheat flour to be 5.5-6.0, inoculating Aspergillus oryzae, and fermenting in a pool to obtain fermented grain koji;
s6, preparing soy sauce mash, namely mixing the prepared fermented grain yeast with salt water with the concentration of 20-25 g/100ml, and then pumping the mixture into a fermentation tank to prepare the soy sauce mash, wherein the weight ratio of the fermented grain yeast to the salt water is 1 (1.5-1.8);
s7, fermenting, namely adding the salt tolerance response reaction substance prepared in the step S4, the lactobacillus fermentation strain and yeast into a fermentation tank, fermenting at 15-20 ℃ for 10 days, heating to 27-30 ℃, fermenting for 60 days, cooling to 20-25 ℃ and fermenting for 10 days to obtain the mature soy sauce mash;
s8, squeezing and sterilizing, namely squeezing the mature soy sauce mash according to a squeezing process to obtain raw soy sauce, filtering the raw soy sauce by diatomite and microporous membranes, sterilizing, transferring into a precipitation tank after sterilizing, standing for storage, precipitating for 30 days, and sub-packaging.
2. The method for producing soy sauce by fermentation with high salt tolerance lactic acid bacteria according to claim 1, wherein: the amino acid composition includes at least two of proline, alanine, aspartic acid, threonine, and tryptophan; in the amino acid composition, the proline is as follows by weight ratio: alanine: aspartic acid: threonine: tryptophan is 1 (0.3-0.8): 0-0.5: (0-0.3).
3. The method for producing soy sauce by fermentation with high salt tolerance lactic acid bacteria according to claim 1, wherein: the lactic acid bacteria in the S2 comprise pediococcus halophilus, micrococcus sojae, lactococcus lactis, pediococcus acidilactici and lactobacillus plantarum, and the pediococcus halophilus is calculated according to the weight ratio: micrococcus soyabean sauce: lactococcus lactis: pediococcus acidilactici: the lactobacillus plantarum is 1 (0.5-1) (0.3-0.5) (0.2-0.5) (0.3-0.8).
4. The method for producing soy sauce by fermentation with high salt tolerance lactic acid bacteria according to claim 1, wherein:
the rotation speed collected by centrifugation in the step S3 is 6000rpm, the time is 10-20 min, and the temperature is 4-10 ℃; the mass ratio of the volume of the PBS buffer to the lactobacillus cells was (50-80) ml/1 g.
5. The method for producing soy sauce by fermentation with high salt tolerance lactic acid bacteria according to claim 1, wherein: the power of ultrasonic crushing in the step S4 is 300-350W, the ultrasonic is performed for 5S, the ultrasonic is stopped for 5S, and the ultrasonic time is 30-50 min; collecting supernatant after ultrasonic crushing, wherein the centrifugal speed is 12000-15000 rpm, the time is 10-15 min, and the temperature is 4-10 ℃; the membrane filtration pore size was 0.22. Mu.m.
6. The method for producing soy sauce by fermentation with high salt tolerance lactic acid bacteria according to claim 1, wherein: the mass ratio of the salt tolerance response reaction substance, the lactobacillus fermentation strain, the yeast and the soy sauce mash is 1 (28-35) (300-500) (1000-12000).
7. The method for producing soy sauce by fermentation with high salt tolerance lactic acid bacteria according to claim 1 or 6, wherein: the lactobacillus fermentation strain comprises at least one of Pediococcus halophilus, micrococcus sojae, lactococcus lactis, pediococcus acidilactici and Lactobacillus plantarum; the yeast is mixed yeast powder of the roux yeast and the globus hystericus, and the roux yeast is prepared from the following materials in percentage by weight: the yeast is (5-6) and (1-2).
8. The method for producing soy sauce by fermentation with high salt tolerance lactic acid bacteria according to claim 1, wherein: the fineness of the diatomite in the S8 is 10# and 95 ℃; the particle size of the microporous membrane is 0.45 mu m; the sterilization temperature is 95 ℃ and the sterilization time is 20-30 min.
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