CN112998260A - Enzyme rich in high-activity enzyme and preparation method thereof - Google Patents
Enzyme rich in high-activity enzyme and preparation method thereof Download PDFInfo
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Images
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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
- A23F3/08—Oxidation; Fermentation
- A23F3/10—Fermentation with addition of microorganisms or enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/06—Treating tea before extraction; Preparations produced thereby
- A23F3/14—Tea preparations, e.g. using additives
-
- 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
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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/175—Rhamnosus
Abstract
The invention discloses a ferment rich in high-activity enzyme and a preparation method thereof, relating to the technical field of food processing; the preparation method of the ferment disclosed by the invention adopts a segmented fermentation technology combining aerobic and anaerobic probiotics to produce 16 fruit and vegetable tea ferments rich in high-activity biological enzymes necessary for human metabolism, wherein 12 of the fruit and vegetable tea ferments are the high-activity biological enzymes participating in TCA circulation and are beneficial to human metabolism; the enzyme product prepared by the invention has the effects of eliminating superoxide anion free radicals, calming nerves, resisting anxiety, relieving depression, helping sleep, promoting alcohol metabolism, enhancing immunity, maintaining intestinal flora balance, promoting digestion, reducing blood pressure and cholesterol, preventing and inhibiting tumors, losing weight and the like, and compared with the existing fruit and vegetable tea enzyme, the enzyme prepared by the invention can more comprehensively provide nutrition for human bodies; the method has the advantages of simple process, convenient operation, pilot plant test and easy implementation and popularization.
Description
Technical Field
The invention relates to the technical field of food processing, and particularly relates to a ferment rich in high-activity enzyme and a preparation method thereof.
Background
Enzymes generally refer to enzymes, which are proteins or RNAs produced by living cells with high specificity and high catalytic activity for their substrates. The chemical nature of an enzyme is protein or RNA, and thus it also has a primary, secondary, tertiary, or even quaternary structure. According to their molecular composition, they can be classified into simple enzymes and conjugated enzymes. Proteins alone are called simplex enzymes; the binding enzyme is composed of an enzyme protein and a cofactor. For example, most hydrolases consist solely of proteins; the flavin mononucleotidase consists of an enzyme protein and a cofactor. The enzyme protein in the combined enzyme is a protein part, the cofactor is a non-protein part, and the enzyme protein and the cofactor have catalytic activity only when the enzyme protein and the cofactor are combined into a whole enzyme. The ferment can also be prepared from animal, plant, and fungus by fermenting with microorganism with or without adjuvant to obtain product containing specific bioactive components (including polysaccharides, oligosaccharides, proteins and polypeptides, amino acids, and vitamins).
At present, although patent and technical literature describe the preparation method of the edible ferment, the problems of unclear effective ingredients, unclear action mechanism and unclear active content still exist, and the existing ferment products contain all active protease participating in TCA circulation at present. The TCA cycle, also known as the citrate cycle or the tricarboxylic acid cycle, is a ubiquitous metabolic pathway in aerobic organisms. The TCA cycle is the final metabolic pathway for carbohydrates, lipids and amino acids, and is also the pivotal junction of carbohydrate, lipid and amino acid metabolic links. Therefore, the preparation of the ferment taking the active protease in the TCA cycle as the main nutrient component has important significance for the development of the ferment industry.
Disclosure of Invention
The present invention aims to provide a ferment rich in high-activity enzyme and a preparation method thereof, so as to solve the problems of the prior art.
In order to achieve the above objects, the present invention provides an enzyme rich in highly active enzymes,
the enzyme comprises: succinic acid thiokinase 240-320U/L, superoxide dismutase 9000-12000U/L and lipase 300-400U/L.
Further, the ferment further comprises: 185U/L of reduced coenzyme I140-.
The invention also provides a preparation method of any enzyme, which comprises the following steps:
(1) first-stage fermentation: adding sugar and water into vegetable juice, fruit juice or tea, mixing, sterilizing, adding yeast activating solution, and fermenting to obtain fermented product;
(2) and (3) secondary fermentation: pasteurizing the fermentation product obtained in the step (1), and adding lactobacillus plantarum activation liquid and bacillus aceticus activation liquid for fermentation to obtain a fermentation product;
(3) three-stage fermentation: and (3) pasteurizing the fermentation product obtained in the step (2), and adding a lactobacillus paracasei activation solution and a lactobacillus rhamnosus activation solution for fermentation to obtain the fruit or vegetable ferment.
Further, the ratio of the vegetable juice, the fruit juice or the tea leaves to the sugar and the water in the first-stage fermentation is 3-4: 0.8-1.2: 0.9-1.5.
Further, the activation method of the yeast activation solution comprises the steps of preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, respectively adding 5-10g of yeast, and activating at 30 ℃ for 30min to prepare the yeast activation solution;
the activating method of the lactobacillus plantarum activating solution comprises the steps of preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, adding 0.1-0.3g of 5000 hundred million CFU/g lactobacillus plantarum, and activating at 32 ℃ for 20-50min to prepare the lactobacillus plantarum activating solution;
the activating method of the acetobacter aceti activating solution comprises the steps of preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, adding 0.1-0.3g of 5000 hundred million CFU/g of acetobacter aceti, and activating at 32 ℃ for 20-50min to prepare the acetobacter aceti activating solution;
the activation method of the lactobacillus paracasei and lactobacillus rhamnosus activation liquid comprises the steps of preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, adding 0.1-0.3g of 1000 hundred million CFU/mL lactobacillus paracasei and lactobacillus rhamnosus, and activating at 32 ℃ for 20-50min to prepare the lactobacillus paracasei and lactobacillus rhamnosus activation liquid.
Further, the specific method for fermenting the yeast activation solution comprises the following steps: adding yeast activating solution, fermenting at 28-32 deg.C and 200r/min under oxygen charging amount of 40LPM for 26 hr, adding water 1.2-1.8kg, adding yeast activating solution, and fermenting at 28-32 deg.C and 200r/min under oxygen charging amount of 40LPM for 16-46 hr.
Further, the specific method for fermenting the lactobacillus plantarum activation solution and the lactobacillus aceticus activation solution comprises the following steps: adding lactobacillus plantarum activating solution and bacillus aceticus activating solution, and fermenting at 30-35 deg.C for 30-60 hr at 200 r/min.
Further, the specific method for fermenting the lactobacillus paracasei activation solution and the lactobacillus rhamnosus activation solution comprises the following steps: adding lactobacillus paracasei activating solution and lactobacillus rhamnosus activating solution, and fermenting at 35-42 deg.C for 30-60 hr at 200 r/min.
Further, the sugar is one or more of honey sugar, white sugar and brown sugar.
The invention discloses the following technical effects: (1) the invention produces 16 enzymes rich in high-activity biological enzymes necessary for human metabolism by probiotic aerobic and anaerobic staged fermentation technologies, wherein 12 enzymes are high-activity biological enzymes participating in TCA circulation and are beneficial to human metabolism; (2) the enzyme product prepared by the invention has the effects of eliminating superoxide anion free radicals, calming nerves, resisting anxiety, relieving depression, helping sleep, promoting alcohol metabolism, enhancing immunity, maintaining intestinal flora balance, promoting digestion, reducing blood pressure and cholesterol, preventing and inhibiting tumors, losing weight and the like, and compared with the existing fruit and vegetable tea enzyme, the enzyme prepared by the invention can more comprehensively provide nutrition for human bodies; (3) the preparation method of the enzyme disclosed by the invention is simple in process, convenient to operate and easy to implement and popularize.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 is a flow chart of a three-stage fermentation process of ferment according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
The yeasts used in the following examples are commercially available products, and Lactobacillus plantarum, Lactobacillus acetobacter, Lactobacillus paracasei and Lactobacillus rhamnosus are isolated and screened special strains by Sichuan province food fermentation industry research and design institute Limited. The fruits or vegetables used in the following examples are all commercially available products.
The preparation method of the fruit, vegetable, tea or traditional Chinese medicine enzyme rich in high-activity enzyme disclosed by the invention is suitable for kiwi fruit, fig, lemon, grape, blueberry, rose, mulberry, sea buckthorn, hair grass root, aloe, wild jujube, sweet osmanthus, pear, green plum, ponkan orange, honeysuckle, cherry, radix stemonae, beetroot root, purple perilla, noni fruit, loquat, pine pollen, blood orange, grapefruit, medlar, tea, edible fungi, bamboo and the like.
The invention adopts the special ferment microbial inoculum for probiotics to produce the ferment with high-activity biological enzyme in a large scale by a subsection fermentation technology combining aerobic and anaerobic processes, and the preparation method of the ferment rich in the high-activity enzyme comprises the following steps:
1. first-stage fermentation:
thawing fruit juice or vegetable juice in advance, weighing 3-4kg of fruit juice or vegetable juice, respectively placing into 10L fermentation tanks, adding 0.8-1.2kg of sugar and 0.9-1.5kg of water, mixing, and sterilizing at 75-85 deg.C for 15-45 min. Preparing 60-100mL 2% glucose solution, sterilizing at 121 deg.C for 15-30min, cooling, adding 5-10g yeast respectively, and activating at 30 deg.C for 30min to obtain yeast activating solution. Pouring a bottle of yeast activating solution into a fermentation tank, uniformly mixing, and then starting fermentation, wherein the fermentation conditions are as follows: fermenting at 28-32 deg.C and 200r/min with oxygen charging amount of 40LPM for 26 hr, adding water 1.2-1.8kg, and adding yeast activating solution. Fermenting at 28-32 deg.C for 16-46 h.
The aerobic fermentation bacteria preparation is mainly characterized by adding value to culture of aroma-enhancing yeast, wherein the main metabolite of yeast added value is superoxide dismutase (SOD) which is a free radical scavenger and can scavenge superoxide anion free radicals, which can lead to lipid peroxidation, human body aging, tissue cell apoptosis acceleration and lesion such as tumor, autoimmune disease, inflammation and the like.
2. And (3) secondary fermentation:
heating the fermentation tank to 75-85 deg.C, pasteurizing for 15-45min, and rapidly cooling. Preparing 60-100mL of 2% glucose solution, sterilizing at 121 deg.C for 15-30min, cooling, adding 0.1-0.3g of 5000 hundred million CFU/g Lactobacillus plantarum, and activating at 32 deg.C for 20-50min to obtain probiotic activating solution. Activating the acetic acid bacillus bacterial liquid at 37 deg.C for 20-50 min. Inoculating activating liquid into the fermentation tank. And (3) starting fermentation after uniformly mixing, wherein the fermentation conditions are as follows: fermenting at 30-35 deg.C for 30-60 hr at 200 r/min.
The two-stage fermentation mainly comprises lactobacillus plantarum multiplication culture and acetobacter multiplication culture, wherein the lactobacillus plantarum uses a metabolite gamma-aminobutyric acid which belongs to a transmitter, has very beneficial effects on brain activities of people, and has the effects of calming nerves, resisting anxiety, relieving depression, helping sleep and promoting alcohol metabolism; the acetobacter is used to consume alcohol generated by a section of fermentation side reaction, so that the alcohol is converted into acetic acid, and the alcohol content is reduced to be less than or equal to 0.5 percent.
3. Three-stage fermentation:
heating to 75-85 deg.C, pasteurizing for 15-45min, and rapidly cooling. Activating Lactobacillus paracasei bacterial liquid at 35-42 deg.C for 30-50min, inoculating 100mL5.85 × 108The fermentation temperature of the CFU/mL lactobacillus paracasei and lactobacillus rhamnosus bacterial liquid is 35-42 ℃, the fermentation time is 30-60h, and the fermentation time is 200 r/min.
The three-stage fermentation is mainly characterized in that lactobacillus paracasei and lactobacillus rhamnosus are added-value culture, wherein the lactobacillus paracasei mainly exists in intestinal tracts of a human body, has the effects of enhancing immunity, maintaining the balance of intestinal flora, promoting digestion and the like, has the effects of reducing blood pressure, reducing cholesterol, preventing cancers, inhibiting tumor growth, enhancing physique, delaying senescence, prolonging life and the like, participates in the digestion function, promotes intestinal peristalsis, and can effectively prevent and treat constipation; the lactobacillus rhamnosus is a kind of colonizing bacterium, has the biological characteristics of acid resistance, bile salt resistance, multiple antibiotic resistance and the like, is one of normal flora of intestinal tract, and can inhibit the growth of harmful bacteria due to the colonizing effect, thereby promoting beneficial bacteria, reducing cholesterol, promoting cell division, regulating intestinal flora, balancing and improving gastrointestinal function, preventing and treating diarrhea, removing toxin, improving the immunity of organism and resisting cancer.
The fruit and vegetable tea enzyme disclosed by the invention contains the following high-activity biological enzymes: reduced coenzyme I (NADH)140-185U/L, acetyl coenzyme A (A-CoA)110-150U/L, Citrate Synthase (CS)25-35U/L, Aconitase (ACO)195-253U/L, isocitrate dehydrogenase (ICD)38-52U/L, Succinate Dehydrogenase (SDH)250-340U/L, Succinate Thiokinase (STK)240-320U/L, alpha-ketoglutarate dehydrogenase (alpha-DHKGC) 155-210U/L, succinyl coenzyme A (SCoA)400-530U/L, Oxalosuccinase (OA)30-40U/L, Fumarase (Fumarase)19-28U/L, Malate Dehydrogenase (MDH)15-28U/L, GTP enzyme KRAS 50-70U/L, and, Oxalyl acetate synthetase 110-150U/L, superoxide dismutase (SOD)9000-12000U/L, and Lipase (Lipase) 300-400U/L.
Wherein, the antioxidant effect of superoxide dismutase (SOD) is obvious; reduced coenzyme I (NADH) is nicotinamide adenine dinucleotide (NAD +) + hydrogen (H), and can be rapidly decomposed into NAD +, hydrogen (H) in vivo and provide energy, so that the composition has the effect of delaying senescence; lipase can play a good role in losing weight. Therefore, the enzyme prepared by the invention has obvious effects of resisting oxidation, resisting aging and losing weight.
The three-stage fermentation process of the ferment of the present invention is shown in FIG. 1.
Example 1 preparation of a fruit and vegetable tea enzyme enriched with highly active enzymes, using kiwi fruit as an example
1.1 one-stage fermentation
Thawing Chinese gooseberry juice in advance, weighing 3.5kg of Chinese gooseberry juice, putting into a 10L fermentation tank, adding 1.0kg of honey and 1.2kg of water, mixing, and sterilizing at 80 deg.C for 30 min. Preparing 80mL of 2% glucose solution, sterilizing at 121 deg.C for 20min, cooling, adding 7g of yeast, respectively, and activating at 30 deg.C for 30min to obtain a yeast activation solution. Pouring a bottle of yeast activating solution into a fermentation tank, uniformly mixing, and then starting fermentation, wherein the fermentation conditions are as follows: 30 ℃, 200r/min, oxygen charging amount of 40LPM, fermenting for 26h, adding 1.5kg of water into the tank, and then adding a part of yeast activating solution. Continuing to ferment for 30h at the temperature of 30 ℃.
1.2 two-stage fermentation
The temperature of the fermenter is raised to 80 deg.C, pasteurized for 30min, and rapidly cooled. Preparing 80mL of 2% glucose solution, sterilizing at 121 deg.C for 20min, cooling, adding 0.2g of 5000 hundred million CFU/g Lactobacillus plantarum, and activating at 32 deg.C for 30min to obtain Lactobacillus plantarum activating solution. The acetic acid bacteria liquid is activated for 30min at 37 ℃. Inoculating activating liquid into the fermentation tank. And (3) starting fermentation after uniformly mixing, wherein the fermentation conditions are as follows: fermenting at 32 ℃ and 200r/min for 45h, and finishing the fermentation.
1.3 three-stage fermentation
The temperature of the fermenter is raised to 80 deg.C, pasteurized for 30min, and rapidly cooled. Activating Lactobacillus paracasei bacterial liquid at 38 deg.C for 40min, inoculating 100ml5.85 × 108The fermentation temperature of the CFU/mL lactobacillus paracasei and lactobacillus rhamnosus bacterial liquid is 37 ℃, the fermentation time is 45h, and the fermentation time is 200 r/min.
1.4 determination of the type of active enzyme and enzyme Activity in Kiwi fruit ferment
Reduced coenzyme I (NADH)171.15U/L, acetyl coenzyme A (A-CoA)135.82U/L, Citrate Synthase (CS)26.85U/L, Aconitase (ACO)206.1U/L, isocitrate dehydrogenase (ICD)42.98U/L, Succinate Dehydrogenase (SDH)310.92U/L, Succinate Thiokinase (STK)270.98U/L, alpha-ketoglutarate dehydrogenase (alpha-KGDHC) 205.66U/L, succinyl-CoA (SCoA)497.09U/L, oxalyl-succinate (OA)35.67U/L, Fumarase (Fumarase)22.07U/L, Malate Dehydrogenase (MDH)21.86U/L, GTP enzyme KRas (KRAS)59.63U/L, oxalyl-acetate synthetase 134.94U/L, superoxide dismutase (SOD)10.66U/mL, Lipase (Lipase) 389.25U/L.
Example 2 preparation of a fruit and vegetable tea enzyme rich in highly active enzyme, using mulberries as an example
2.1 first stage fermentation
Thawing the mulberry juice in advance, weighing 3kg of mulberry juice, putting into a 10L fermentation tank, adding 0.8kg of white granulated sugar and 0.9kg of water, mixing uniformly, and sterilizing at 75 ℃ for 15 min. Preparing 60mL of 2% mulberry sugar solution, sterilizing at 121 ℃ for 15min, cooling, respectively adding 5g of yeast, and activating at 30 ℃ for 30min to obtain a yeast activation solution. Pouring a part of yeast activating solution into a fermentation tank, uniformly mixing, and then starting fermentation, wherein the fermentation conditions are as follows: fermenting at 28 deg.C and 200r/min with oxygen charging amount of 40LPM for 26 hr, adding 1.2kg of water, and adding one part of yeast activating solution. The fermentation was continued at 28 ℃ for 16 h.
2.2 two-stage fermentation
The temperature of the fermenter was raised to 75 deg.C, pasteurized for 15min, and rapidly cooled. Preparing 60mL of 2% mulberry sugar solution, sterilizing at 121 ℃ for 15min, cooling, adding 0.1g of 5000 hundred million CFU/g of lactobacillus plantarum, and activating at 32 ℃ for 20min to prepare lactobacillus plantarum activating solution. The acetic acid bacteria liquid is activated for 20min at 37 ℃. Inoculating activating liquid into the fermentation tank. And (3) starting fermentation after uniformly mixing, wherein the fermentation conditions are as follows: fermenting at 30 ℃ for 30h at a speed of 200r/min, and finishing the fermentation.
2.3 three-stage fermentation
The temperature in the fermenter was raised to 75 deg.C, pasteurized for 15min, and rapidly cooled. Activating Lactobacillus paracasei bacterial liquid at 35 deg.C for 30min, inoculating 100ml5.85 × 108The fermentation temperature of the CFU/mL lactobacillus paracasei and lactobacillus rhamnosus bacterial liquid is 35 ℃, the fermentation time is 30h, and the fermentation time is 200 r/min.
2.4 determination of the type of active enzyme and enzyme Activity in Mulberry enzyme
Reduced coenzyme I (NADH)163.98U/L, acetyl coenzyme A (A-CoA)134.47U/L, Citrate Synthase (CS)27.49U/L, Aconitase (ACO)214.49U/L, isocitrate dehydrogenase (ICD)48.26U/L, Succinate Dehydrogenase (SDH)268.21U/L, Succinate Thiokinase (STK)292.00U/L, alpha-ketoglutarate dehydrogenase (alpha-KGDHC) 193.72U/L, succinyl-CoA (SCoA)429.58U/L, oxalyl-succinate (OA)34.86U/L, Fumarase (Fumarase)22.13U/L, Malate Dehydrogenase (MDH)21.81U/L, GTP enzyme KRas (KRAS)57.77U/L, oxalyl-acetate synthetase 133.23U/L, superoxide dismutase (SOD)11.89U/mL, Lipase (Lipase) 365.50U/L.
Example 3 preparation of a fruit and vegetable tea enzyme enriched with highly active enzymes, using Roughhaired grass root as an example
3.1 first stage fermentation
Thawing the root juice in advance, weighing 4kg of the root juice, putting into a 10L fermentation tank, adding 1.2kg of brown sugar and 1.5kg of water, mixing, and sterilizing at 85 deg.C for 45 min. Preparing 100mL of 2% glucose solution, sterilizing at 121 deg.C for 30min, cooling, adding 10g of yeast, respectively, and activating at 30 deg.C for 30min to obtain a yeast activation solution. Pouring a part of yeast activating solution into a fermentation tank, uniformly mixing, and then starting fermentation, wherein the fermentation conditions are as follows: fermenting at 32 deg.C and 200r/min with oxygen charging amount of 40LPM for 26 hr, adding water 1.8kg, and adding yeast activating solution. The fermentation was continued for 46h at 32 ℃.
3.2 two-stage fermentation
The temperature of the fermenter was raised to 85 deg.C, pasteurized for 45min, and rapidly cooled. Preparing 100mL of 2% glucose solution, sterilizing at 121 deg.C for 30min, cooling, adding 0.3g of 5000 hundred million CFU/g Lactobacillus plantarum, and activating at 32 deg.C for 50min to obtain Lactobacillus plantarum activating solution. The acetic acid bacteria liquid is activated for 50min at 37 ℃. Inoculating activating liquid into the fermentation tank. And (3) starting fermentation after uniformly mixing, wherein the fermentation conditions are as follows: fermenting at 35 deg.C for 60h at 200 r/min.
3.3 three-stage fermentation
The temperature in the fermenter was raised to 85 deg.C, pasteurized for 45min, and rapidly cooled. Activating Lactobacillus paracasei bacterial liquid at 42 deg.C for 50min, inoculating 100ml5.85 × 108The fermentation temperature of the CFU/mL lactobacillus paracasei and lactobacillus rhamnosus bacterial liquid is 42 ℃, the fermentation time is 60h, and the fermentation time is 200 r/min.
3.4 determination of the type of active enzyme in the Roughhaired grass root enzyme and enzyme Activity
Reduced coenzyme I (NADH)164.83U/L, acetyl coenzyme A (A-CoA)128.73U/L, Citrate Synthase (CS)31.73U/L, Aconitase (ACO)206.66U/L, isocitrate dehydrogenase (ICD)47.14U/L, Succinate Dehydrogenase (SDH)255.30U/L, Succinate Thiokinase (STK)255.28U/L, alpha-ketoglutarate dehydrogenase (alpha-KGDHC) 174.18U/L, succinyl-CoA (SCoA)415.77U/L, oxalyl-succinate (OA)36.21U/L, Fumarase (Fumarase)21.15U/L, Malate Dehydrogenase (MDH)23.29U/L, GTP enzyme KRas (KRAS)58.45U/L, oxalyl-acetate synthetase 118.83U/L, superoxide dismutase (SOD)10.31U/mL, Lipase (Lipase) 346.27U/L.
Example 4 preparation of a fruit and vegetable tea enzyme enriched with highly active enzymes, using tea as an example
4.1 one-stage fermentation
Weighing 4kg of fresh honeysuckle tea, putting into a 10L fermentation tank, adding 1.1kg of honey, white granulated sugar and brown sugar and 1.3kg of water, mixing uniformly, and sterilizing at 85 deg.C for 45 min. Preparing 100mL of 2% glucose solution, sterilizing at 121 deg.C for 30min, cooling, adding 10g of yeast, respectively, and activating at 30 deg.C for 30min to obtain a yeast activation solution. Pouring a part of yeast activating solution into a fermentation tank, uniformly mixing, and then starting fermentation, wherein the fermentation conditions are as follows: fermenting at 32 deg.C and 200r/min with oxygen charging amount of 40LPM for 29 hr, adding water 1.8kg, and adding yeast activating solution. The fermentation was continued at 29 ℃ for 46 h.
4.2 two-stage fermentation
The temperature of the fermenter was raised to 82 deg.C, pasteurized for 45min, and rapidly cooled. Preparing 100mL of 2% glucose solution, sterilizing at 121 deg.C for 30min, cooling, adding 0.3g of 5000 hundred million CFU/g Lactobacillus plantarum, and activating at 32 deg.C for 40min to obtain Lactobacillus plantarum activating solution. The acetic acid bacteria liquid is activated for 50min at 37 ℃. Inoculating activating liquid into the fermentation tank. And (3) starting fermentation after uniformly mixing, wherein the fermentation conditions are as follows: fermenting at 35 deg.C for 50 hr/min at 200 r/min.
4.3 three-stage fermentation
The temperature in the fermenter was raised to 85 deg.C, pasteurized for 45min, and rapidly cooled. Activating Lactobacillus paracasei bacterial liquid at 42 deg.C for 50min, inoculating 100ml5.85 × 108The fermentation temperature of the CFU/mL lactobacillus paracasei and lactobacillus rhamnosus bacterial liquid is 42 ℃, the fermentation time is 60h, and the fermentation time is 200 r/min.
4.4 determination of the type of active enzyme in tea ferment and enzyme Activity
Reduced coenzyme I (NADH)153.45U/L, acetyl coenzyme A (A-CoA)133.45U/L, Citrate Synthase (CS)27.64U/L, Aconitase (ACO)226.89U/L, isocitrate dehydrogenase (ICD)50.06U/L, Succinate Dehydrogenase (SDH)262.25U/L, Succinate Thiokinase (STK)302.60U/L, alpha-ketoglutarate dehydrogenase (alpha-KGDHC) 189.92U/L, succinyl-CoA (SCoA)458.73U/L, oxalyl-succinate (OA)33.51U/L, Fumarase (Fumarase)22.02U/L, Malate Dehydrogenase (MDH)20.96U/L, GTP enzyme KRas (KRAS)68.26U/L, oxalyl-acetate synthetase 130.14U/L, superoxide dismutase (SOD)10.49U/mL, Lipase (Lipase) 304.41U/L.
Example 5 enzyme stability experiment
If the enzyme product is improperly processed, the phenomena of precipitation, browning, acidification and the like can occur in the product in shelf life, so that the product quality is reduced, the enzyme activity in the enzyme is reduced and the like, and therefore, stability experiments are performed on the products (examples 1 to 4) prepared by the preparation method disclosed by the invention.
5.1 enzyme activity Change assay
After being stored for 1 year under the condition of shade and normal temperature, the enzyme activities of various enzymes in the four enzymes are measured, and compared with the enzyme activities in the examples 1 to 4, the enzyme activities are attenuated, and the attenuation is less than or equal to 20%.
5.2 sensory evaluation of enzyme products
10 professional chefs, 10 food processing professionals and 10 laymen were invited to form a sensory evaluation panel, and the four enzymes prepared in examples 1 to 4 were subjected to sensory evaluation, and then the four enzymes stored for 300 days were subjected to sensory evaluation, and the evaluation results showed that the taste was softer and more uniform, the taste was pure, and the color was more clear and transparent after storage for 300 days.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (9)
1. An enzyme enriched in a highly active enzyme, comprising: succinic acid thiokinase 240-320U/L, superoxide dismutase 9000-12000U/L and lipase 300-400U/L.
2. The ferment of claim 1, wherein the ferment further comprises: 185U/L of reduced coenzyme I140-.
3. The method for preparing ferment according to claim 1 or 2, comprising the following steps:
(1) first-stage fermentation: adding sugar and water into vegetable juice, fruit juice or tea, mixing, sterilizing, adding yeast activating solution, and fermenting to obtain fermented product;
(2) and (3) secondary fermentation: pasteurizing the fermentation product obtained in the step (1), and adding lactobacillus plantarum activation liquid and bacillus aceticus activation liquid for fermentation to obtain a fermentation product;
(3) three-stage fermentation: and (3) pasteurizing the fermentation product obtained in the step (2), and adding a lactobacillus paracasei activation solution and a lactobacillus rhamnosus activation solution for fermentation to obtain the fruit or vegetable ferment.
4. The method of claim 3, wherein the ratio of the vegetable juice, fruit juice or tea leaves to sugar and water in the first stage of fermentation is 3-4: 0.8-1.2: 0.9-1.5.
5. The method according to claim 3, wherein the activation of the yeast activation solution is performed by preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, adding 5-10g of yeast, respectively, and activating at 30 ℃ for 30min to obtain the yeast activation solution;
the activating method of the lactobacillus plantarum activating solution comprises the steps of preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, adding 0.1-0.3g of 5000 hundred million CFU/g lactobacillus plantarum, and activating at 32 ℃ for 20-50min to prepare the lactobacillus plantarum activating solution;
the activating method of the acetobacter aceti activating solution comprises the steps of preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, adding 0.1-0.3g of 5000 hundred million CFU/g of acetobacter aceti, and activating at 32 ℃ for 20-50min to prepare the acetobacter aceti activating solution;
the activation method of the lactobacillus paracasei and lactobacillus rhamnosus activation liquid comprises the steps of preparing 60-100mL of 2% glucose solution, sterilizing at 121 ℃ for 15-30min, cooling, adding 0.1-0.3g of 1000 hundred million CFU/mL lactobacillus paracasei and lactobacillus rhamnosus, and activating at 32 ℃ for 20-50min to prepare the lactobacillus paracasei and lactobacillus rhamnosus activation liquid.
6. The method according to claim 3, wherein the specific method for fermenting the yeast activation solution is: adding yeast activating solution, fermenting at 28-32 deg.C and 200r/min under oxygen charging amount of 40LPM for 26 hr, adding water 1.2-1.8kg, adding yeast activating solution, and fermenting at 28-32 deg.C and 200r/min under oxygen charging amount of 40LPM for 16-46 hr.
7. The preparation method according to claim 3, wherein the specific method for fermenting the lactobacillus plantarum activation solution and the lactobacillus aceticus activation solution is as follows: adding lactobacillus plantarum activating solution and bacillus aceticus activating solution, and fermenting at 30-35 deg.C for 30-60 hr at 200 r/min.
8. The preparation method of claim 3, wherein the specific method for fermenting the lactobacillus paracasei activation solution and the lactobacillus rhamnosus activation solution is as follows: adding lactobacillus paracasei activating solution and lactobacillus rhamnosus activating solution, and fermenting at 35-42 deg.C for 30-60 hr at 200 r/min.
9. The method of claim 3, wherein the sugar is one or more of honey sugar, white sugar and brown sugar.
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Application publication date: 20210622 |