CN105105119A - Method for preparing fruit enzyme by fermenting based on inoculated strain - Google Patents

Abstract

This case involves a method for fermenting and preparing fruit enzymes based on inoculated strains, including: selecting materials, peeling and slicing, adjusting sugar content, sterilizing, adding modified fermented liquid, fermenting and filtering; wherein, the modified fermented liquid is fermented naturally The liquid is inoculated with modified strains selected from at least one of Aspergillus oryzae, saccharomyces, Streptococcus thermophilus and Lactobacillus bulgaricus. In this case, beneficial bacteria were added under the original process conditions, so that microbial enzymes developed in the direction of design, no longer limited to the selection of enzyme raw materials, and can maximize the antioxidant activity and enzyme activity of microbial enzymes; adding modified bacteria After planting, the changing rules of various indicators of the enzyme and the resulting impact can be presented during the experiment, which provides certain data support and theoretical basis for the research and development of functional microbial enzyme products and the realization of industrial production.

Description

A method for fermenting and preparing fruit enzymes based on inoculated strains

technical field

The invention relates to a method for preparing enzymes, in particular to a method for fermenting and preparing fruit enzymes based on inoculated strains.

Background technique

Enzyme is a healthy food that people are keen on in recent years. It can be seen everywhere in TV commercials or online marketing. In fact, it is the "enzyme" that people are very familiar with. Almost all organs, tissues and cells in the human body need to rely on the catalytic reaction of enzymes and the supply of energy to maintain their power and health. Enzymes can promote the metabolism of the human body, making us happy physically and mentally; they can promote blood circulation, eliminate toxins in the body, and purify our blood system; they can strengthen the digestion and absorption of our gastrointestinal tract and enhance our physical fitness; they can regulate the acidity in our body Alkaline balance is beneficial to the detoxification of the liver; it can also promote the repair of damaged cells and activate cells.

At the beginning of the 20th century, enzymes became popular in Japan, and then they were introduced in Taiwan, Singapore, Malaysia, South Korea, and the United States, and set off wave after wave of enzyme fever wherever they went, and the response was extremely enthusiastic. With the advancement of the times, human beings attach great importance to health, and enzyme-related industries have developed rapidly, and enzyme food has attracted more and more attention. In the process of food fermentation, carbohydrates are continuously consumed to reduce the fat content. Therefore, human beings have invested a long time cost and high economic cost to ferment the food and then eat it not only to change the taste, but also to be more nutritious. academic considerations.

Microbial enzymes refer to functional fermented foods rich in vitamins and minerals produced by fermenting one or more fruits and vegetables as raw materials. During the whole fermentation process of the enzyme, the microorganisms make various changes in the raw materials through their own metabolism, and generate new biologically active substances and enzymes without affecting the original nutrients. These new bioactive substances include a variety of functional nutrients such as phenolic substances, organic acids and sugars, and have made certain contributions to human health. Phenolic substances mainly include anthocyanins, flavonoids, tannins, lignin, catechins, styrene, coumarin, flavonols, tannins, phenolic acids, etc.; organic acids mainly include malic acid, succinic acid , pyruvic acid, gallic acid, etc.

In recent years, microbial enzymes have become popular all over the world, and functional products of microbial enzymes have emerged in an endless stream. Its functions of anti-oxidation, antibacterial and anti-inflammatory, promoting metabolism, improving immunity, whitening and anti-aging are highly sought after by people. The production process of microbial enzymes is constantly updated, however, most of the research is done based on the traditional fermentation process. This microbial enzyme, which is naturally fermented by traditional fermentation techniques, is easily affected by conditions such as microorganisms in the environment, fermentation process, and seasons. It is difficult to artificially control product quality, thus affecting its development and utilization. At present, most of the research at home and abroad is based on natural fermentation, studying the metabolites produced during the fermentation process, the fermentation mechanism, etc., and there is no relevant research showing that adding specific beneficial bacteria in the early stage of fermentation will affect the fermentation. impact, and whether such a solution is feasible.

Contents of the invention

For the deficiencies of the prior art, the purpose of the present invention is to explore the process of developing new functional microbial enzyme products, to provide data support and theoretical basis for realizing industrialized production, to adopt artificial inoculation of bacterial classification, to its antioxidant activity and enzyme And so on to carry out in-depth tracking and monitoring, so that the fermentation of the product is under control and develops in the expected direction.

In this case, fruit enzymes such as apple enzymes, pear enzymes or citrus enzymes were used as the research object. A certain amount of Aspergillus oryzae, yeast, Lactobacillus acidophilus and Lactobacillus bulgaricus were inoculated at the initial stage of fermentation. Track and monitor its antioxidant activity and its changing law, enzyme activity and changing law. Various aspects such as strain control of microbial enzymes, functional component analysis and product quality control, as well as comprehensive development and utilization of enzymes are reflected.

The key to this case study is to select 4 beneficial fermentation strains, artificially inoculate a certain proportion of the strains on the basis of the original enzyme production, and study the changes in the fermentation process. These 4 strains included Aspergillus oryzae, Saccharomyces, Streptococcus thermophilus and Lactobacillus bulgaricus. During the whole fermentation process, these four kinds of bacteria play different roles, but they are not independent individuals, but complement each other. Therefore, the grasp of the proportion of the bacteria is very important, and it also determines the fermentation of the enzyme. quality.

The summary of the technical solution of the present invention is as follows:

A method for fermenting and preparing fruit enzymes based on inoculated strains, comprising: selecting materials, peeling and slicing, adjusting sugar content, sterilizing, adding modified fermented liquid, fermenting and filtering; wherein, the modified fermented liquid is fermented naturally The liquid is inoculated with modified strains selected from at least one of Aspergillus oryzae, saccharomyces, Streptococcus thermophilus and Lactobacillus bulgaricus.

Preferably, the method for fermenting fruit enzymes based on inoculated strains, wherein the modified strains are selected from at least two of Aspergillus oryzae, Saccharomyces, Streptococcus thermophilus and Lactobacillus bulgaricus.

Preferably, the method for fermenting fruit enzymes based on inoculated strains, wherein the modified strains are selected from at least three of Aspergillus oryzae, Saccharomyces, Streptococcus thermophilus and Lactobacillus bulgaricus.

Preferably, the method for fermenting and preparing fruit enzymes based on inoculated strains, wherein the modified strains are a combination of Aspergillus oryzae, Saccharomyces, Streptococcus thermophilus and Lactobacillus bulgaricus.

Preferably, in the method for fermenting fruit enzymes based on inoculated strains, the inoculum amount of the modified strains is 2-12%. (Note: According to the default expression habit of those skilled in the art, the % here represents the volume percentage, and the % used when expressing the inoculum amount herein is a volume percentage unless otherwise specified)

Preferably, in the method for fermenting fruit enzymes based on inoculated strains, the inoculum amount of each strain in the modified strains is 0.5-3%.

Preferably, the method for fermenting and preparing fruit enzymes based on inoculated strains, wherein, in the modified strains, the inoculation amount of Aspergillus oryzae is 1.5%, the inoculum amount of yeast is 1.0%, and the thermophilic chain The inoculation amount of cocci was 1.0%, and that of Lactobacillus bulgaricus was 1.0%.

Preferably, the method for fermenting and preparing fruit enzymes based on inoculated strains, wherein, specifically includes:

Step 1) Filter the fruit enzymes after natural fermentation for 1 month to obtain natural fermentation liquid, sterilize the natural fermentation liquid at 115°C for 10 minutes, cool to room temperature, and inoculate and activate the natural fermentation liquid separately under the ultra-clean workbench Aspergillus oryzae, saccharomycetes, Streptococcus thermophilus and Lactobacillus bulgaricus in the described modified strains were fermented at room temperature for 1 month, and filtered to obtain the modified fermented liquid;

Step 2) Select fresh fruits, clean them with sterile water under aseptic conditions, dry them naturally in an aseptic operating table, peel them, and slice them for later use;

Step 3) adding fruit and white granulated sugar to a sterilized glass bottle in a mass ratio of 1:1;

Step 4) adding the modified fermented liquid into a glass bottle, sealing, storing in a dark place, fermenting at room temperature for 30-90 days, and filtering to obtain the fruit enzyme.

The beneficial effects of the present invention are:

1) Transition from natural fermentation to artificial inoculation

In recent years, microbial enzyme food has become popular all over the world. Most of the research at home and abroad tends to be natural fermentation, that is, without adding any additional strains. Such traditional process methods often have no way to control the quality and fermentation time of the enzyme. In this case, the modified strains are added under the original process conditions to make the microbial enzyme develop in the direction of design. It is no longer limited to the selection of enzyme raw materials and can maximize Maximize the improvement of the antioxidant activity and enzyme activity of microbial enzymes.

2) Can track and monitor the enzyme fermentation process

Regarding the tracking and monitoring of the fermentation process of microbial enzymes, the total phenol content, reducing power and hydroxyl radicals, superoxide anion radicals, ABTS free radicals, DPPH free radical scavenging rate, superoxide dismutase, Systematic detection of amylase, lipase, protease, cellulase, total acid, total sugar, alcohol and pH. After adding the modified strains of Aspergillus oryzae, yeast, Streptococcus thermophilus and Lactobacillus bulgaricus, the changes of various indicators of the enzyme and the impact can be presented during the experiment, which is useful for the research and development of functional microbial enzymes It provides certain data support and theoretical basis for the realization of industrial production; at the same time, it further solves the problems of microbial enzyme strain control, functional components and product quality control.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments, so that those skilled in the art can implement it with reference to the description.

Aspergillus oryzae, yeast, Streptococcus thermophilus and Lactobacillus bulgaricus are mature commercially available products, and their culture methods also belong to the prior art, and can be easily obtained by those skilled in the art, so there is no need to provide their preservation information in this case.

Example 1:

Activation of bacteria:

(1) Aspergillus oryzae medium

Comprehensive potato medium (PDA): 1L of 20% potato, 20g of glucose, 3g of KH ₂ PO ₄ , 1.5g of MgSO ₄ ·7H ₂ O, 8mg of VitanibB1Trace, 20g of agar, natural pH, autoclaved at 115°C for 25min.

(2) Yeast medium

10°Bé wort agar medium: Dilute the fermented beer raw material (without adding hops) to 10 Berlin, add agar 15g/L, dissolve and aliquot, and autoclave at 121°C for 15 minutes.

(3) Medium for Streptococcus thermophilus

Yeast extract 1%, glucose 1.5%, beef extract 0.3%, peptone 0.5%, CaCO ₃ 1.0%, Tween 801mL, agar 2%, pH 6.5-7.0, autoclaved at 115°C for 25min.

(4) Lactobacillus bulgaricus medium

Yeast extract 1%, glucose 1.5%, beef extract 0.3%, peptone 0.5%, CaCO ₃ 1.0%, Tween 801mL, agar 2%, pH 6.5-7.0, autoclaved at 115°C for 25min.

Prepare solid medium according to the formula of the above-mentioned medium. Inoculate the surviving Aspergillus oryzae, saccharomyces, Streptococcus thermophilus and Lactobacillus bulgaricus on the corresponding solid medium respectively, and cultivate them under suitable conditions (Aspergillus oryzae, yeast: 25～28°C; Streptococcus thermophilus , Lactobacillus bulgaricus: 43 ℃), activated for 2 to 3 generations, so that the strain's vitality reaches the strongest.

Example 2:

Production of modified fermentation broth:

Natural fermentation broth (that is, no additional strains added)→inoculation of pure strains→fermentation culture→mixing according to different volume ratios→modified fermentation broth.

Example 3:

Enzyme production process:

Select raw material (fruit) → peel → slice → adjust sugar content → kill microorganisms and miscellaneous bacteria in the air → activate bacteria → add modified fermentation liquid → ferment → obtain enzyme liquid containing pulp → filter.

Example 4:

Enzyme production process:

Clean fresh apples with sterile water under sterile conditions, dry them naturally in a sterile operating table, peel them, and slice them for later use; add apples and white sugar in a mass ratio of 1:1 to a sterilized glass bottle; add the modified fermentation liquid into a glass bottle, seal it, put it in a dark place, and ferment at room temperature for 30 to 90 days. In order to avoid the contamination of miscellaneous bacteria, all operations in the process of making enzymes are carried out under sterile conditions.

Example 5:

Add single strain fermentation test:

Filter the apple enzyme after natural fermentation for 1 month to obtain natural fermented liquid. Divide the natural fermentation broth evenly into 5 parts, select one part as the blank control, sterilize the remaining 4 parts at 115°C for 10 minutes, cool to room temperature, and inoculate Aspergillus oryzae and yeast separately under the ultra-clean workbench , Streptococcus thermophilus and Lactobacillus bulgaricus. 5 parts of fermented liquids with different treatment methods were sealed and fermented at room temperature for 1 month.

Embodiment 6:

Sensory evaluation:

Select 7 people as representatives of the appraisal team, under the same environment and conditions, score the sensory evaluation of the prepared enzyme, including the color, smell and mouthfeel of the enzyme; ~100 points (including 80), relatively good scores are 70-80 points (including 70), acceptable scores are 60-70 points (including 60), and completely unacceptable scores are less than 60 points. The total score is 100 points, which is obtained by adding the scores of the three categories. The specific proportions are: 30% for color, 25% for smell, and 45% for taste.

Embodiment 7:

Effects of different proportions of Aspergillus oryzae inoculum on the sensory evaluation of enzymes:

The inoculation amount of Streptococcus thermophilus and Lactobacillus bulgaricus is preferably 2% to 3%, and the quality and taste of the fermented product are the best when the strain ratio is 1:1 or 2:1, and the commonly used ratio is 1:1. The inoculation amount of yeast, Streptococcus thermophilus and Lactobacillus bulgaricus selected in the experiment was 3.0%, and the inoculation amount of Aspergillus oryzae was changed to 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%. After 30 days of fermentation, samples were taken to obtain a fermented liquid containing pulp and all enzymes, and the enzyme supernatant was obtained by filtration. The enzyme supernatant was taken as an experimental sample for sensory evaluation.

Embodiment 8:

Effects of different proportions of yeast inoculum on sensory evaluation of enzymes:

The inoculum amount of Aspergillus oryzae is 2.0%, the inoculum amount of Streptococcus thermophilus and Lactobacillus bulgaricus is 3.0%, and the inoculum amount of yeast is changed to 0.5%, 1.0%, and 1.5% respectively without changing other process conditions , 2.0%, 2.5%, 3.0%. After 30 days of fermentation, samples were taken to obtain a fermented liquid containing pulp and all enzymes, and the enzyme supernatant was obtained by filtration. The enzyme supernatant was taken as an experimental sample for sensory evaluation.

Embodiment 9:

Effects of different proportions of Streptococcus thermophilus inoculum on sensory evaluation of enzymes:

The inoculation amount of Aspergillus oryzae is 2.0%, the inoculum amount of saccharomyces is 1.0%, the inoculum amount of Lactobacillus bulgaricus is 3.0%, and the inoculum amount of Streptococcus thermophilus is changed to 0.5% respectively without changing other process conditions , 1.0%, 1.5%, 2.0%, 2.5%, 3.0%. After 30 days of fermentation, samples were taken to obtain a fermented liquid containing pulp and all enzymes, and the enzyme supernatant was obtained by filtration. The enzyme supernatant was taken as an experimental sample for sensory evaluation.

Example 10:

Effects of different proportions of Lactobacillus bulgaricus inoculum on sensory evaluation of enzymes:

The inoculum amount of Aspergillus oryzae is 2.0%, the inoculum amount of saccharomyces is 1.0%, the inoculum amount of Streptococcus thermophilus is 1.5%, and the inoculum amount of Lactobacillus bulgaricus is changed to 0.5% respectively without changing other process conditions , 1.0%, 1.5%, 2.0%, 2.5%, 3.0%. After 30 days of fermentation, samples were taken to obtain a fermented liquid containing pulp and all enzymes, and the enzyme supernatant was obtained by filtration. The enzyme supernatant was taken as an experimental sample for sensory evaluation.

Example 11:

Orthogonal Design of Strain Proportion

According to the single factor test, four factors were selected: the inoculation amount of Aspergillus oryzae (A), the inoculum amount of yeast (B), the inoculum amount of Streptococcus thermophilus (C) and the inoculum amount of Lactobacillus bulgaricus (D), and each factor took 3 Orthogonal experiment was carried out, and the factor levels are shown in Table 1.

Table 1 Factor level table

Tab.1 Orthogonal factor level table

Orthogonal test results and analysis are shown in Table 2.

Table 2 Orthogonal test results and analysis

Tab.2 Result and analysis for thogonal results

As can be seen from Table 2, the main factor affecting the sensory evaluation of enzymes is the inoculation amount of Aspergillus oryzae, followed by Streptococcus thermophilus, then Lactobacillus bulgaricus, and finally yeast. And when the inoculation amount of Aspergillus oryzae was 1.5%, that of yeast was 1.0%, that of Streptococcus thermophilus was 1.0%, and that of Lactobacillus bulgaricus was 1.0%, the sensory evaluation scores of microbial enzymes produced by mixed fermentation were the highest. Carry out verification test by the analysis result of orthogonal test, the inoculum amount of Aspergillus oryzae is 1.5%, the inoculum amount of saccharomyces is 1.0%, the inoculum amount of Streptococcus thermophilus is 1.0%, the inoculum amount of Lactobacillus bulgaricus is at 1.0%, With other process conditions unchanged, the sensory evaluation score of the obtained enzyme solution was 92.3, which was better than any group in the above-mentioned tests.

Example 12:

Enzyme Antioxidant Activity

The total phenolic content, reducing power and scavenging ability of superoxide free radicals, DPPH free radicals, ABTS free radicals and hydroxyl free radicals in the experimental group increased with the increase of concentration. The scavenging ability of free radicals and ABTS free radicals is better than that of the control group (blank experiment); the enzymes of the control group have a particularly good scavenging ability for hydroxyl radicals; generally speaking, the free radical scavenging ability of the experimental group is higher than that of the control group Clearance.

Example 13:

Changes of enzyme antioxidant activity

With the prolongation of the fermentation time, the trend of each antioxidant active ingredient in the fermentation liquid of the experimental group and the control group was different, and the changes were complex and changeable, depending on whether the strains were added and the fermentation raw materials themselves. In general, the total phenolic content, reducing power and free radical scavenging ability of enzymes generally showed an upward trend.

(1) Comparing the strains added with apple enzyme with those without strains

On the 60th day of fermentation, the total phenol content increased by 15%, the reducing power increased by 1.8%, the superoxide anion radical scavenging ability increased by 36.55%, the hydroxyl radical scavenging ability decreased by 4.27%, and the DPPH free radical scavenging rate Increased by 59%, ABTS free radical scavenging capacity increased by 3.10%.

(2) Compared with pear enzyme added strains and no added strains

On the 60th day of fermentation, the total phenol content increased by 10.17%, the reducing power increased by 4.90%, the superoxide anion radical scavenging ability increased by 5.44%, the hydroxyl radical scavenging ability increased by 2.67%, DPPH free radical scavenging The rate increased by 39.78%, and the ABTS free radical scavenging ability increased by 6.84%.

(3) Comparing the strains added with citrus enzymes with those without strains

On the 60th day of fermentation, the total phenol content increased by 30.85%, the reducing power increased by 17.57%, the superoxide anion radical scavenging ability increased by 7.46%, the hydroxyl radical scavenging ability increased by 6.26%, and the DPPH free radical scavenging rate Increased by 38.08%, ABTS free radical scavenging ability increased by 4.76%.

Example 14:

Enzyme Activity Variation Law

With the prolongation of fermentation time, the change of enzyme activity did not show a single increasing law, but increased and decreased. This also shows that enzymes are not as people often say, the longer the better, but you should choose to drink at the right time.

(1) SOD enzyme activity

The control group reached the maximum on the 30th day of the test, with a maximum of 296U/mL; the experimental group reached the maximum on the 75th day of the test, with a maximum of 268U/mL.

(2) Amylase activity

The control group reached the maximum on the 45th day of the test, the maximum was 46U/mL; the experimental group reached the maximum on the 60th day of the test, the maximum was 76U/mL.

(3) Lipase activity

The control group reached the maximum at the 30th day of the test, with a maximum of 9.6 U/mL; the experimental group reached the maximum at the 75th day of the test, with a maximum of 6.8 U/mL.

(4) Protease activity

The control group reached the maximum on the 15th day of the test, with a maximum of 655U/mL; the experimental group reached the maximum on the 45th day of the test, with a maximum of 890U/mL.

(5) Cellulase activity

The control group reached the maximum on the 45th day of the test, the maximum was 47.9U/mL; the experimental group reached the maximum on the 30th day of the test, the maximum was 63.7U/mL.

The activity of microbial enzymes differs greatly between artificial inoculation of strains and natural fermentation. Compared with no added strains, on the 90th day of fermentation, SOD enzyme activity decreased by 22.39%, amylase activity increased by 50%, lipase activity decreased by 69.49%, protease activity increased by 85.71%, cellulose Enzyme activity increased by 54.19%.

Example 15:

Variation rules of enzyme total acid, total sugar, alcohol content and pH

When the fermentation time was 30 days, the total acid content of the ferment solution showed an upward trend, but after 30 days, the total acid content decreased and finally stabilized. In the first 15 days of fermentation, the total acid content accumulated rapidly, the total sugar content decreased significantly, a large amount of alcohol was produced, and the pH value dropped sharply. After 15 days, it enters the slow fermentation stage. Compared with no strain added, on the 90th day of fermentation, the total acid content increased by 24.14%, the total sugar content decreased by 12.5%, the alcohol content increased by 16.67%, and the pH decreased by 5%.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details and embodiments shown here, without departing from the general concept defined by the claims and their equivalents.

Claims (8)

1. A method for fermenting and preparing fruit enzymes based on inoculated strains, comprising: material selection, peeling and sectioning, adjusting sugar content, sterilizing, adding modified fermented liquid, fermentation and filtration; wherein, the modified fermented liquid is in The natural fermentation broth is inoculated with modified strains, and the modified strains are selected from at least one of Aspergillus oryzae, yeast, Streptococcus thermophilus and Lactobacillus bulgaricus. 2. the method for fermenting and preparing fruit ferment based on inoculated bacterial classification as claimed in claim 1, is characterized in that, described modified bacterial classification is selected from at least in Aspergillus oryzae, saccharomycete, Streptococcus thermophilus and Lactobacillus bulgaricus two kinds. 3. the method for fermenting and preparing fruit ferment based on inoculated bacterial classification as claimed in claim 1, is characterized in that, described modified bacterial classification is selected from at least in Aspergillus oryzae, saccharomycete, Streptococcus thermophilus and Lactobacillus bulgaricus three kinds. 4. the method for fermenting and preparing fruit ferment based on inoculation strain as claimed in claim 1, is characterized in that, described modified strain is the combination of aspergillus oryzae, saccharomycete, streptococcus thermophilus and Lactobacillus bulgaricus four . 5. The method according to any one of claims 1-4, wherein the inoculum amount of the modified strain is 2-12%. 6. The method for fermenting and preparing fruit enzymes based on inoculated strains as claimed in claim 4, characterized in that, the inoculation amount of each strain in the modified strains is 0.5 to 3%. 7. the method for fermenting and preparing fruit ferment based on inoculated bacterial classification as claimed in claim 6, is characterized in that, in described modified bacterial classification, the inoculum size of Aspergillus oryzae is 1.5%, and the inoculum size of saccharomycete is 1.0% , the inoculation amount of Streptococcus thermophilus was 1.0%, and that of Lactobacillus bulgaricus was 1.0%. 8. the method for fermenting and preparing fruit ferment based on inoculated bacterial classification as claimed in claim 7, is characterized in that, specifically comprises: Step 1) Filter the fruit enzymes after natural fermentation for 1 month to obtain natural fermentation liquid, sterilize the natural fermentation liquid at 115°C for 10 minutes, cool to room temperature, and inoculate and activate the natural fermentation liquid separately under the ultra-clean workbench Aspergillus oryzae, saccharomycetes, Streptococcus thermophilus and Lactobacillus bulgaricus in the described modified strains were fermented at room temperature for 1 month, and filtered to obtain the modified fermented liquid; Step 2) Select fresh fruits, clean them with sterile water under aseptic conditions, dry them naturally in an aseptic operating table, peel them, and slice them for later use; Step 3) adding the fruit and white sugar to the sterilized glass bottle in a mass ratio of 1:1; Step 4) adding the modified fermented liquid into a glass bottle, sealing, storing in a dark place, fermenting at room temperature for 30-90 days, and filtering to obtain the fruit enzyme.