CN110786500B - Method for preparing perilla sauce by mixed fermentation method - Google Patents
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- CN110786500B CN110786500B CN201910815163.8A CN201910815163A CN110786500B CN 110786500 B CN110786500 B CN 110786500B CN 201910815163 A CN201910815163 A CN 201910815163A CN 110786500 B CN110786500 B CN 110786500B
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- 235000004347 Perilla Nutrition 0.000 title claims abstract description 83
- 235000015067 sauces Nutrition 0.000 title claims abstract description 76
- 238000000855 fermentation Methods 0.000 title claims abstract description 72
- 230000004151 fermentation Effects 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 26
- 244000124853 Perilla frutescens Species 0.000 title claims description 106
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- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims abstract description 11
- 235000004348 Perilla frutescens Nutrition 0.000 claims description 30
- 238000011081 inoculation Methods 0.000 claims description 21
- 239000002994 raw material Substances 0.000 claims description 18
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- 239000000725 suspension Substances 0.000 claims description 11
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- 108091005804 Peptidases Proteins 0.000 abstract description 47
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 47
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- 108010073682 nattokinase Proteins 0.000 abstract description 43
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- 102000006995 beta-Glucosidase Human genes 0.000 abstract description 4
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- 241000229722 Perilla <angiosperm> Species 0.000 abstract 7
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- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 4
- 244000063299 Bacillus subtilis Species 0.000 description 3
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- 235000018102 proteins Nutrition 0.000 description 3
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- 238000002835 absorbance Methods 0.000 description 2
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 2
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- XMGQYMWWDOXHJM-UHFFFAOYSA-N limonene Chemical compound CC(=C)C1CCC(C)=CC1 XMGQYMWWDOXHJM-UHFFFAOYSA-N 0.000 description 2
- 229960004488 linolenic acid Drugs 0.000 description 2
- 235000013557 nattō Nutrition 0.000 description 2
- LVHLZMUFIYAEQB-UHFFFAOYSA-N perilla ketone Chemical compound CC(C)CCC(=O)C=1C=COC=1 LVHLZMUFIYAEQB-UHFFFAOYSA-N 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
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- RUMOYJJNUMEFDD-SNVBAGLBSA-N (R)-(+)-Perillaldehyde Natural products CC(=C)[C@H]1CCC(C=O)=CC1 RUMOYJJNUMEFDD-SNVBAGLBSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 244000000231 Sesamum indicum Species 0.000 description 1
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- 235000010208 anthocyanin Nutrition 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000004141 dimensional analysis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000018927 edible plant Nutrition 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000001510 limonene Nutrition 0.000 description 1
- 229940087305 limonene Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000013433 optimization analysis Methods 0.000 description 1
- RUMOYJJNUMEFDD-UHFFFAOYSA-N perillyl aldehyde Chemical compound CC(=C)C1CCC(C=O)=CC1 RUMOYJJNUMEFDD-UHFFFAOYSA-N 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002975 protease activity determination Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000013077 scoring method Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A23L27/60—Salad dressings; Mayonnaise; Ketchup
-
- 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
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Agronomy & Crop Science (AREA)
- Botany (AREA)
- Seeds, Soups, And Other Foods (AREA)
Abstract
The invention discloses a method for preparing perilla sauce by a mixed fermentation method. The invention utilizes the Bacillus belgii SN-14 which produces nattokinase, protease and beta-glucosidase and has excellent performance and the elegant mucor actinomucor which produces protease to grow fast, and the fermented perilla sauce has strong ester fragrance, and the two are combined together, thereby not only leading the nattokinase, the protease and the beta-glucosidase which are produced by the perilla sauce to have rich content and moderately enzymolyze macromolecules such as protein, fiber and the like, but also neutralizing the bitter taste of bean pulp and perilla which are produced by single strain fermentation, leading the final product to have rich nutrition, not only bringing new active ingredients, but also leading special nutrient ingredients, active ingredients and flavor substances in the perilla cake to be completely retained in the perilla sauce, and having good flavor and texture. The finally obtained perilla sauce product is superior to the common traditional bean sauce food in the aspects of seasoning and increasing flavor.
Description
Technical Field
The invention relates to the technical field of food processing, in particular to a method for preparing perilla sauce by a mixed fermentation method.
Background
Perilla frutescens, named as common perilla frutescens, is an annual herbaceous plant of Perilla in Labiatae, also called as perilla frutescens, is an important and traditional medicinal and edible plant in China all the time, and is one of 60 special dual-purpose oil crops published in the Ministry of health in China. Perilla frutescens is treasure, leaves and stems of the perilla frutescens mainly contain terpenoids, phenols, fatty acids and flavonoids, perilla frutescens is rich in oil, the oil content can be as high as 40% -45%, the alpha-linolenic acid content in the fatty acids can be as high as 65%, the protein content of the perilla frutescens is increased to about 35% after the perilla frutescens is subjected to a degreasing process, and the perilla frutescens is rich in anthocyanin, limonene, perilla ketone, perilla aldehyde and other special nutritional and active ingredients. The main residue of the perilla seeds after the oil extraction process is called perilla cake, at present, the cake left after the oil extraction is only used as a raw material of products such as bread, extruded protein and the like, and most perilla cake is only used as feed for low-value utilization. In recent years, although the research heat of relevant industries at home and abroad on perilla series products is not reduced, the related industries only carry out crude extraction research on perilla cake protein, and further intensive development and research are not carried out. In fact, the perilla cake is rich in nutrition, contains high crude fiber and crude protein besides rich alpha-linolenic acid, so that sauce and seasoning prepared by fermentation may have unique taste and smell. However, many perilla cakes on the market are made into feed at low price. At present, the perilla frutescens sauce products on the market are few, the research and development of the perilla frutescens sauce at home and abroad are also in a shallow layer, the perilla frutescens cake, bean pulp, soybeans and the like are subjected to single-bacterium fermentation, most of the strains are aspergillus oryzae, mucor and the like, and the perilla frutescens cake has poor taste under the traditional fermentation condition, cannot give full play to the unique flavor of the perilla frutescens cake and is easily burnt. In addition, few functional bacteria are utilized for fermentation. Therefore, how to better exert the special fresh scent of the perilla without covering the taste and make the taste soft, mellow and rich is a common problem in the current development of perilla sauce products.
Based on the development condition of perilla series products researched and developed at present and the value and the characteristics of perilla, the project sends mucor elegans CICC3118 producing protease and beleis Bacillus SN-14 producing nattokinase (natural good medicine for preventing and treating cardiovascular and cerebrovascular diseases) and protease (digestion-aiding) (the strain is sent to a China center for type culture preservation and is classified and named as Bacillus subtilis SN-14, the preservation number is CCTCC M2017134, the address is Wuhan university in Wuhan, china) to be mixed and fermented according to a certain proportion to produce perilla cake and bean pulp raw materials which are rich in nattokinase and protease and do not cover the special fresh fragrance of perilla and can make the taste of the perilla soft and mellow.
Disclosure of Invention
Aiming at the defects of the existing invention materials, the invention provides a method for preparing perilla frutescens sauce by a mixed fermentation method, which is rich in nattokinase and protease, has the special faint scent of perilla frutescens, and is soft and mellow in taste.
In order to achieve the purpose, the invention is realized by the following technical scheme: the method for preparing the perilla sauce by the mixed fermentation method comprises the following steps:
(1) Pre-treating raw materials, namely screening and grinding bean pulp and perilla hemp cake pulp, mixing according to a certain amount, adding water into a mixture, soaking and standing for 1h until the mixture is fully absorbed by the raw materials, wherein the material-water ratio is not less than 1:4, the absorption degree is held to be conglobate, water is separated out, the loose is dispersed, flour accounting for 1-5% of the total mass of the materials is scattered on the surface of the material amount, and then steaming and sterilizing at 121 ℃ for 20 min;
(2) Seed liquid culture:
1) Spore suspension of actinomucor elegans CICC3118 was prepared: picking up mucor hyphae stored on a PDA slant, inoculating the mucor hyphae to a PDA plate culture medium, culturing at 28 ℃ for 72h, washing off spores by using sterile normal saline, and storing the obtained spore suspension at 4 ℃ for later use;
2) B, preparing a Bacillus belgii SN-14 seed solution: the liquid seed culture medium is 10-15g/L glucose, 5-10g/L yeast extract, 10-15/L beef extract, 5-10g/L NaCl, pH 7.4-7.6, 2-5 rings of Bacillus belezii SN-14 bacteria are selected by an inoculating loop and inoculated into the liquid seed culture medium, and the liquid seed culture medium is cultured for 16-18h under the conditions of 37 ℃ and 180r/min to obtain Bacillus belezii SN-14 seed liquid;
3) After the raw material mixture is cooled to room temperature, inoculating the prepared spore suspension of the actinomucor elegans CICC3118 (which is a commercial strain) into the raw material, controlling the temperature at 30 ℃ to perform shake cultivation and fermentation for 24 hours, then inoculating the seed solution of the Bacillus beibeilesensis, controlling the temperature at 30 ℃ to perform after-fermentation, and keeping the temperature until the strong fragrance is volatilized;
(4) Adding salt: adding salt according to the amount of 1-3% of the mass of the sauce.
The ratio of the soybean meal to the perilla frutescens cake meal is 5.5-6.5:3.5-4.5, and mixing.
The volume ratio of spore suspension of the actinomucor yawazekii CICC3118 to strains of the Bacillus belgii SN-14 seed liquid is 2.5-3:1, the total inoculation amount of the two seed liquids is 0.4-0.42% of the total mass of the materials.
The Bacillus subtilis (Bacillus velezensis) is Bacillus subtilis SN-14 with the preservation number: CCTCC M2017134. The applicant has previously patented this strain and was the strain screened in the inventors' laboratory.
Compared with the prior art, the invention utilizes the Bacillus belgii SN-14 which produces nattokinase and protease and beta-glucosidase with excellent performance and the fermented perilla sauce which produces protease and has strong ester fragrance, and combines the two together, so that the nattokinase, the protease and the beta-glucosidase which are produced by the perilla sauce are rich, macromolecules such as protein and fiber are properly enzymolyzed, the bitter taste of bean pulp and perilla brought by single strain fermentation can be neutralized, the final product has rich nutrition, not only can bring new active ingredients, but also can completely retain special nutrient ingredients, active ingredients and flavor substances in the perilla sauce, and the flavor and the texture are both good. The finally obtained perilla sauce product is superior to the common traditional bean sauce food in the aspects of seasoning and increasing flavor. Meanwhile, the high-value utilization of the perilla seed cakes can be realized.
Detailed Description
Example (b): the present embodiment takes western style smoked ham as an example of detection by a method for predicting the shelf life of food through dimensional analysis.
1.1 points of operation
(2) The key points of the operation
1) Pre-treating raw materials, namely screening and grinding bean pulp and perilla hemp cake pulp, mixing a certain amount (the bean pulp powder accounts for 60 percent of the total amount, the perilla hemp cake pulp accounts for 40 percent of the total amount), adding water (the material-water ratio should be not less than 1:4), soaking and standing for 1h until the raw materials are fully absorbed, wherein the absorption degree is to hold the raw materials into a mass, water is separated out, the mass is properly dispersed, filling 10-30 percent of flour with the volume in a mixing material bottle, scattering 1-5 percent of flour of the mixing material on the surface of the raw materials, and then steaming and sterilizing at the high temperature of 121 ℃ for 20 min;
2) Seed liquid culture: a spore suspension of mucor was prepared as follows: picking up mucor hyphae stored on a PDA inclined plane, inoculating the mucor hyphae to a PDA plate culture medium, culturing for 72h at 28 ℃, washing off spores by using sterile normal saline, and storing the obtained spore suspension at 4 ℃ for later use; b, preparing a Bacillus belgii seed solution: liquid seed culture medium: 10-15g/L glucose, 5-10g/L yeast extract, 10-15/L beef extract, 5-10g/L NaCl, pH 7.4-7.6, inoculating 2-5 rings of Bacillus beilaisi SN-14 bacteria selected by an inoculating loop into a liquid seed culture medium, and culturing at 37 ℃ for 16-18h at 180r/min to obtain SN-14 seed liquid.
3) After the raw material mixture is cooled to room temperature, the prepared mucor elegans CICC3118 spore suspension is inoculated into the raw material according to a certain strain proportion, the temperature is controlled at 30 ℃ for shake cultivation and fermentation for 24 hours, then the bacillus belgii SN-14 is inoculated according to a certain strain proportion, the temperature is controlled for after-fermentation, and the temperature is kept until the strong fragrance is volatilized;
4) Adding salt: adding salt according to the amount of 1-3% of the sauce mass, or determining the salt adding amount according to the requirement of the final actual production
1.2 protease Activity determination method
Protease activity was measured using Folin-phenol reagent colorimetry.
Definition of enzyme activity: the amount of enzyme required for 1min to decompose casein to produce 1. Mu.g of tyrosine in 1g of sample at 40 ℃ and pH7.0 is 1 protease activity unit (U).
1.3 Nattokinase activity determination method
And (3) activity determination: adding 1.4mL Tris-HCl (50 mmol/L, pH 7.8) buffer solution and 0.4mL fibrinogen solution (7.2 mg/mL) into a test tube, incubating at 37 ℃ for 5min, adding 0.1mL thrombin (20U/mL), incubating at 37 ℃ for 10min to form an artificial thrombus, adding 0.1mL sample to be tested, incubating at 37 ℃ for 60min, adding 2mL trichloroacetic acid (0.2 mol/L) solution, standing for 20min to terminate the reaction, centrifuging at 13000r/min for 10min, and taking the supernatant and measuring the absorbance at 275nm wavelength.
Definition of enzyme activity: the amount of enzyme required for an increase in absorbance of 0.01 at 27nm per minute was defined as 1 unit of fibrin-degrading enzyme activity.
1.4 sensory evaluation of Perilla frutescens sauce
The relevant indexes of a single perilla sauce product are integrated, and a perilla sauce sensory quality scoring method is formulated according to the aspects of color, smell, taste, tissue stability and the like of the product, namely, after the perilla sauce is fermented and prepared according to the color, smell, taste and tissue stability, 5 sensory evaluators are asked to conduct sensory evaluation on the perilla sauce product according to the scoring rules in the table 1, and finally, the final evaluation is obtained according to the scoring of the 5 sensory evaluators. (see Table 1 for detailed rules of evaluation)
TABLE 1 Perilla sauce sensory score
Case 2
2.1 Single factor test results
2.1.1 Effect of different strain proportions on the enzyme activity values and sensory scores of nattokinase and protease generated in perilla sauce
The method has the advantages that the defects of low activity value of produced nattokinase and protease enzyme and over-thick and heavy odor when the perilla sauce is fermented by a single strain of Bacillus belgii SN-14 are overcome, and the sauce is prepared by mixed fermentation with the actinomucor elegans CICC3118, so that the enzyme activity values of the nattokinase and the protease in a finished product are greatly improved, the over-thick and heavy odor is synthesized, the sauce is softer and has faint scent, and the quality of the perilla sauce is improved. The influence of the proportions of different bacteria on the enzyme activity values of nattokinase and protease generated in perilla sauce is shown in table 2, the enzyme activity values of nattokinase and protease are gradually increased along with the increase of the strain proportion, but when the strain proportion is 3:1, compared with the aspects of fragrance, texture, taste and the like after fermentation for 48 hours, the fragrance of soybean meal and the fragrance of perilla sesame cake meal can be kept, no bad smell is generated, unique light sweet smell of spleen ester is generated, the sauce fragrance overflows, the viscosity is moderate, the enzyme activity values of nattokinase and protease are on a medium level, and 3:1 can be used as the optimal strain proportion and further subjected to fermentation research. Therefore, in the response surface analysis optimization experiment, the strain ratio of the actinomucor elegans CICC3118 and the Bacillus belgii SN-14 is set as 2:1, 3:1 and 4:1.
TABLE 2 influence of different strain ratios on the enzyme activity values of nattokinase and protease in perilla sauce
Sensory Scoring
Nine groups are set up for sensory evaluation under different strain proportions. After fermenting for 48 hours, carrying out sensory evaluation on the finally fermented perilla sauce finished product, and grading from the aspects of color, smell, taste and tissue form to obtain the average color value of the perilla sauce of 15; the odor value was 13; the mouth feel value is 15; the tissue morphology value was 6.5.1:1 to 2:3 are not rich enough, and the ester flavor is not completely volatilized, and compared with the previous groups, in the group 3, 2, 3.
2.1.2 Effect of different amounts of inoculum on the Activity of Nattokinase and protease in Perilla sauce and sensory evaluation
The effect of different inoculum sizes on the enzyme activity values of nattokinase and protease produced in perilla sauce is shown in table 3. Under 5 different inoculation amounts, when the inoculation amount is more than 0.3%, the enzyme activity values of the produced nattokinase and the protease in the finished perilla sauce product are obviously and greatly increased, because the number of strains in the finished perilla sauce product is increased along with the increase of the inoculation amount, the utilization rate of nutrient substances in the raw materials is also increased, and further the secretion of enzyme systems such as the protease, the nattokinase and the like is increased. In addition, in 5 groups of different inoculum sizes, the 0.2% and 0.3% inoculum groups were bland in taste, had only a weak suavena scent, had poor consistency and had strong fluidity. The group with the inoculation amount of 0.35 percent and the group with the inoculation amount of 0.4 percent have rich and sweet taste, are thick and fragrant in sauce, mellow, stronger in ester fragrance and free of other bad smells, and the unique perilla hemp smell overflows more than other groups, is moderately sticky and free of impurities; however, the group inoculated with 0.45% had a heavier taste, a slightly sour taste and a burnt taste, and was more nasty than the other group 4. Through comparison of odor, taste, texture, enzyme activity values of protease, nattokinase and the like, the inoculation amount of 0.4 percent can be optimized, and further research and optimization are carried out. Therefore, in the response surface analysis optimization experiment, the inoculation amounts of the actinomucor yawayanensis CICC3118 and the Bacillus belgii SN-14 are 0.3%, 0.4% and 0.5%.
TABLE 3 Effect of different inoculum sizes on the values of nattokinase and protease activities in Perilla frutescens sauce
Sensory Scoring
At different inoculum sizes, five groups were set up for sensory evaluation. The strain proportion is 3:1, fermentation is carried out for 48 hours, sensory evaluation is carried out on the finished perilla sauce product after final fermentation, grading is carried out on the aspects of color, smell, taste and tissue form, and the average color value of the perilla sauce is 16; an odor value of 14; the mouth feel value is 15; the tissue morphology value was 7.5. The results show that, in 5 groups of different inoculum sizes, the 0.2% and 0.3% inoculum size groups were light in taste, only weak suavena aroma was produced, the consistency was poor, and the fluidity was strong. The group with the inoculation amount of 0.35 percent and the group with the inoculation amount of 0.4 percent have rich and sweet taste, are thick and fragrant in sauce, mellow, stronger in ester fragrance and free of other bad smells, and the unique perilla hemp smell overflows more than other groups, is moderately sticky and free of impurities; however, the group inoculated with 0.45% had a heavier taste, a slightly sour taste and a burnt taste, and was more nasty than the other group 4.
2.1.3 influence of different fermentation time on the enzyme activity value and sensory score of nattokinase and protease generated in perilla sauce
The fermentation time is an important influence factor in the sauce making and fermentation process, and hyphae cannot fully grow when the fermentation time is too long, so that the quality and the smell of the perilla sauce are greatly influenced, and the enzyme activity values of nattokinase and protease in the perilla sauce are also influenced; however, if the fermentation time is too long, other mixed bacteria are easily stained in the fermentation process, so that the smell, the texture and the taste of the perilla sauce are damaged, and the perilla sauce can compete with high-quality fermentation strains for nutrient substances in raw materials, so that the generation of nattokinase and protease is greatly reduced, and the control of the fermentation time is particularly important. As can be seen from Table 4, the influence of different fermentation time periods on the enzyme activity values of nattokinase and protease in the perilla sauce is that the enzyme activity values of the produced nattokinase and protease gradually increase along with the increase of the fermentation time period. However, after 6 groups of different fermentation time periods, the finished perilla sauce has obvious difference in taste. The taste was too light and the posture was too thin for the 36h group; compared with the group 44h, the group 40h has similar smell, the color is brownish but glossy, but after the group 40h is close and smelled, the perilla flavor of the group 40h is more natural and rich, the sauce flavor is rich, the ester flavor is mellow, and no other bad smell exists, but after the fermentation time is prolonged to 48h, the taste is aggravated, and the longer the fermentation time is (when the fermentation time is longer than 48 h), the taste becomes sharp and has sour, and the perilla flavor is covered by other bad smells similar to natto. Finally, the enzyme activity value of protease and nattokinase and the like are combined, and the fermentation time of 40h can be used as the preferable option of the fermentation of the perilla jam. Therefore, in the response surface analysis optimization experiment, the fermentation time can be set to be 36h, 40h and 44h.
TABLE 4 influence of different fermentation durations on the enzyme activity values of nattokinase and protease in perilla frutescens paste
Sensory scoring
Six groups were set up for sensory evaluation at different fermentation durations. The strain proportion is that 3:1 is firstly used, the inoculation amount is 0.4 percent, sensory evaluation is carried out on the finished perilla sauce product after final fermentation, and grading is carried out on the aspects of color, smell, taste and tissue form, so that the average color value of the perilla sauce is 16; odor value of 15; the mouth feel value is 15; the tissue morphology value was 7. After 6 groups of different fermentation time periods, the differences of the taste of the finished perilla sauce are obvious. The taste was too light and the posture was too thin for the 36h group; compared with the group 44h, the group 40h has similar smell, the color is brownish but glossy, but after the smell is close and fine, the taste of the perilla herb in the group 40h is more natural and full-bodied, the sauce fragrance is rich, the ester fragrance is mellow, no other bad smell exists, but after the fermentation time is prolonged to 48h, the taste is aggravated, and the longer the fermentation time (when the fermentation time is more than 48 h), the taste is more pungent and sour, and the perilla herb smell is covered by other bad smell similar to natto. Case 3 response surface optimization analysis
3.1 determination of test points
On the basis of a single-factor test result, 3 factors of the strain proportion, the inoculation amount and the fermentation time of the mucor elegans CICC3118 and the Bacillus belgii SN-14 are used as self-variable factors, the optimal extraction process of the nattokinase content and the protease content is optimized by a response surface method, and the response surface test Design and data analysis are carried out by Design-Expert 8.0.6 software.
3.2 response surface test level factors and results
A three-factor three-level response surface analysis test is carried out by applying a center combination test design principle according to Box-Behnken and through a strain proportion A, an inoculation amount B and a fermentation duration C. The levels of response surface test factors and results are shown in tables 5 and 6.
TABLE 5 response surface test factor levels and codes
Table 6 response surface test design and results
3.3 response surface test results and analysis of variance thereof
The experimental data were analyzed using Desihn-Expert 8.0.6 software to obtain the quadratic polynomial regression equation:
R1＝46.80-1.5A-0.5B+3.5C-1.75AB-0.25AC-1.25BC-8.53A 2 -6.03B 2 - 6.03C 2
R2＝39.00-1.75A+0.62B+2.88C-0.5AB-1.0AC+1.75BC-4.63A 2 -3.38 B 2 -3.87C 2
wherein R1 and R2 are respectively nattokinase content and protease content; A. b and C respectively correspond to the strain proportion, the inoculation amount (%) and the fermentation time (h) of the actinomucor elegans CICC3118 and the Bacillus belgii SN-14.
3.3.4 influence of Strain proportion, inoculum size and fermentation duration on Nattokinase content
The F value is used for checking the significance of the influence of each variable on the response value in the test, when the P value is smaller, the significance degree of the corresponding variable is higher, and when the mismatching term P is more than 0.05, the mismatching term P is not significant. From the results of the analysis of variance in Table 7, the model was found to be very different from F =100.92, p < 0.0001, and the mismatching term p =0.7510>0.05, indicating that the model is significant. And the colony ratio of the actinomucor elegans CICC3118 and the Bacillus belgii SN-14 and the influence of the fermentation time of the perilla sauce on the enzyme activity content of the nattokinase are obvious (P)<0.05 The influence of the inoculation amount on the enzyme activity content of the nattokinase is not obvious (P)>0.05). Therefore, the influence degree of the three factors on the enzyme activity content of the nattokinase is C>A>B. Furthermore, since R 2 ＝0.9924，Adj R 2 ＝0.9825，Pre R 2 =0.9618, the variance difference is very small and close to 1, which not only shows good fitting degree, but also can be used for analyzing and predicting the optimum extraction process of the nattokinase content. For the interaction, only the interaction of the ratio of the strains of the actinomucor elegans CICC3118 and the Bacillus belgii SN-14 and the fermentation time does not have obvious influence on the enzyme activity content of the nattokinase (P)>0.05 But the interaction of the strain proportion, the inoculation amount and the fermentation time has obvious influence on the enzyme activity content of the nattokinase (P)<0.05)。
TABLE 7 analysis of variance of nattokinase content response surface fitting regression equation
* Significant difference, p<0.05; * Marked by extreme difference, p<0.01；R 2 ＝0.9924,Adj R 2 ＝0.9825,Pre R 2 ＝0.9618
3.5 Effect of Strain proportion, inoculum size and fermentation duration on protease content
From the results of the analysis of variance in Table 8, it can be seen that the differences between F =94.85, p < 0.0001 in the model are very significant, and the mismatching term p =0.7022>0.05, indicating that the model is significant. The proportion of the strains of the actinomucor elegans CICC3118 and Bacillus belgii SN-14 and the duration of fermentation have a significant influence on the protease content (P)<0.05 Whereas the effect of the inoculum size on the protease content was not significant (P)>0.05). Therefore, the influence degree of the three factors on the protease content is C>A>B. Due to R 2 ＝0.9919，Adj R 2 ＝0.9814，Pre R 2 =0.9553, the variance difference is very small and close to 1, which indicates that the fitting degree is good, so the model can be used for analyzing and predicting the optimal extraction process of the enzyme activity content of the protease. For the interaction, the strain ratio and fermentation duration of Mucor delbrueckii CICC3118 and Bacillus belgii SN-14, and the effect of the interaction of the inoculum size and fermentation duration on the protease content were significant (P)<0.05 But the interaction of strain proportion and inoculum size had no significant effect on protease content (P)>0.05)。
TABLE 8 analysis of variance of the protease content surface fitting regression equation
* Significant difference, p<0.05; * Marked by extreme difference, p<0.01；R 2 ＝0.9919,Adj R 2 ＝0.9814,Pre R 2 ＝0.9553
3.6 sensory evaluation
Performing sensory evaluation on 17 groups of perilla frutescens sauce subjected to final response surface analysis and optimized fermentation, and grading from the aspects of color, smell, taste and tissue form to obtain the average color value of the perilla frutescens sauce of 17; odor value of 16.5; the mouth feel value is 16; the tissue morphology value was 7.5. The 17 groups of perilla frutescens sauce have small overall difference, bright color, little difference in taste, flavor and tissue form, but the finished products are all perilla frutescens with strong fragrance and refreshing ester fragrance.
By using Design-Expert 8.0.6 software, the optimal results of the nattokinase content and the protease content in the perilla sauce can be obtained under the common influence of the strain proportion, the inoculation amount (%) and the fermentation time (h): the strain proportion is 2.86, the inoculation amount is 0.41%, the fermentation time is 41.38h, and under the condition, the nattokinase content predicted by the model is 47.2691Fu/g, and the protease content is 39.8112u/g. Then carrying out sensory evaluation on the finally fermented perilla frutescens sauce, and grading from the aspects of color, smell, taste and tissue form to obtain the average color value of the perilla frutescens sauce of 16.3; odor value of 16.3; the mouth feel value is 16.3; the tissue morphology value was 7.5. The results show that the integrated maturity of the Mixi GUAN adopted in the experiment is very high, and the scores of color, smell and taste are good, so that the appearance formed by mixed fermentation in the experiment is uniform brown and glossy, the surface has no black spots, the special aromatic smell and taste of purple perilla, the purple sweet taste is strong, the tissue is partially rough, but the stability is good. In general, the perilla sauce produced in the batch has good sensory quality and can satisfy most consumers.
The fermentation conditions and the optimal formula of the mixed-bacteria fermented flavored perilla frutescens sauce are finally determined by adopting mucor elegans CICC3118 and Bacillus belvesii SN-14 to carry out mixed-bacteria fermentation on bean pulp and perilla frutescens cake pulp, taking the contents of protease and nattokinase as detection indexes and assisting sensory evaluation, and observing the content change and the change of sensory properties of the indexes in the mixed-bacteria fermentation process of the perilla frutescens sauce. The final results show that: after the soybean meal and the perilla seed meal are subjected to mixed fermentation, the contents of protease and nattokinase in finished perilla sauce products have obvious growth trends under different strain proportion, inoculation amount and fermentation time, when the strain proportion of the actinomucor elegans CICC3118 to the Bacillus belgii SN-14 is 2.86, the inoculation amount is 0.41%, temperature-controlled fermentation is carried out for 41.38 hours, the protease content reaches 39.8112u/g, the nattokinase content reaches 47.2691Fu/g, and the perilla sauce finished perilla seed meal and soybean meal are subjected to mixed fermentation according to the formula and have bright color and mellow taste, so that the feasibility of analyzing and optimizing the formula of the mixed fermentation flavored perilla seed sauce by using the response surface method is high.
The above embodiments are merely specific examples of the present invention, and it is obvious that the present invention is not limited to the above embodiments. The invention is within the scope of protection as long as the method concept and technical scheme of the invention are adopted to carry out insubstantial improvement or the concept and technical scheme of the invention are directly applied to other occasions without improvement.
Claims (2)
1. A method for preparing perilla sauce by a mixed fermentation method is characterized by comprising the following steps: the method comprises the following steps:
(1) Pre-treating raw materials, namely screening and grinding bean pulp and perilla hemp cake pulp, mixing, adding water into a mixture, soaking and standing for 1h until the mixture is fully absorbed by the raw materials, wherein the material-water ratio is not less than 1:4, the absorption degree is based on holding to form a cluster, water is separated out, and the cluster is loosened, scattering flour accounting for 1-5% of the total mass of the materials on the surface of the materials, and then steaming and sterilizing at 121 ℃ for 20 min;
(2) Seed liquid culture:
1) Spore suspension of actinomucor elegans CICC3118 was prepared: picking up mucor hyphae stored on a PDA slant, inoculating the mucor hyphae to a PDA plate culture medium, culturing at 28 ℃ for 72h, washing off spores by using sterile normal saline, and storing the obtained spore suspension at 4 ℃ for later use;
2) B, preparing a Bacillus belgii SN-14 seed solution: the liquid seed culture medium is 10-15g/L glucose, 5-10g/L yeast extract, 10-15/L beef extract, 5-10g/L NaCl, pH 7.4-7.6, 2-5 rings of Bacillus belezii SN-14 bacteria are selected by an inoculating loop and inoculated into the liquid seed culture medium, and the liquid seed culture medium is cultured for 16-18h under the conditions of 37 ℃ and 180r/min to obtain Bacillus belezii SN-14 seed liquid;
(3) After the raw material mixture is cooled to room temperature, inoculating the prepared mucor elegans CICC3118 spore suspension into the raw material, controlling the temperature at 30 ℃ to carry out shake culture and fermentation for 24 hours, then inoculating Bacillus belgii seed liquid, controlling the temperature at 30 ℃ to carry out after-fermentation, and keeping the temperature until the strong fragrance is volatilized;
(4) Adding salt: adding salt according to the amount of 1-3% of the mass of the sauce;
the Bacillus belgii is Bacillus belgii SN-14 with the preservation number: CCTCC M2017134;
the volume ratio of the spore suspension of the actinomucor elegans CICC3118 to the strain of the Bacillus belgii SN-14 seed solution in the step (3) is 3:1, and the total inoculation amount of the two seed solutions is 0.35 percent or 0.41 percent of the total mass of the materials.
2. The method for preparing perilla sauce by using the mixed fermentation method according to claim 1, wherein the method comprises the following steps: the ratio of the soybean meal to the perilla frutescens cake meal is 5.5-6.5: mixing at a mass ratio of 3.5-4.5.
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