CN112869141A - Compound lactobacillus crabapple enzyme and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of edible processing, and particularly relates to a compound lactobacillus crabapple enzyme and a preparation method thereof. The preparation method comprises the steps of mixing the crab apple juice with water, adding auxiliary materials, uniformly mixing, inoculating lactobacillus plantarum, lactobacillus bulgaricus and streptococcus thermophilus, performing primary fermentation and secondary fermentation, and performing filling sterilization or aseptic filling to obtain the finished product of the lactobacillus fermented crab apple enzyme. At the moment, the crab apple enzyme is bright orange yellow, the crab apple smell is rich, and the taste is slightly sour and has no impurities. Animal experiments prove that the crab apple enzyme has the capability of improving the intestinal flora of mice and the anti-depression capability.

Description

Compound lactobacillus crabapple enzyme and preparation method thereof

Technical Field

The invention belongs to the technical field of deep processing of food, and particularly relates to a compound lactobacillus crabapple enzyme and a preparation method thereof.

Background

Malus spectabilis (Calopyllum inophyllum L.) is originally produced in China and distributed in northwest, north China, northeast and south of Yangtze river as plants of Rosaceae and Malus. Fruits and seeds are rich in oil, the color of the peel is bright red and dazzling, the pulp is yellow and white, and fresh food is sour, sweet, fragrant and crisp, and has astringent taste due to the fact that the fruit and seeds contain a large amount of tannin substances. The crabapple fruit contains a large amount of nutrients such as saccharides, multiple vitamins, organic acids, etc., can supplement nutrients required by human body, enhance resistance to diseases, has the functions of promoting the production of body fluid to quench thirst, invigorating spleen to arrest diarrhea, and can treat dyspepsia, food stagnation, abdominal distention, hemorrhoid, etc.

Disclosure of Invention

The invention provides a compound lactobacillus crabapple enzyme and a preparation method thereof, finds that the lactobacillus fermented crabapple enzyme has the functions of improving the intestinal flora of mice and resisting depression, and researches and discovers the composition of specific compound lactobacillus to improve the application value of crabapple fruits and ensure the improvement of the intestinal flora of mice and the realization of the depression resistance.

The malus spectabilis ferment prepared by fermenting malus spectabilis fruits through lactic acid bacteria can improve the mouthfeel with obvious astringency. The finished product of the crab apple enzyme is bright orange yellow, has strong smell of crab apple, and has slightly sour and impurity-free mouthfeel. The economic value of the crab apple is improved, and the crab apple enzyme has multiple biological functions.

The technical scheme of the invention is as follows:

firstly, the preparation method of the compound lactobacillus crabapple enzyme comprises the following steps: squeezing fructus Mali Pumilae, adding sugar, performing primary fermentation at 35-39 deg.C for 3-5d, performing secondary fermentation at 25-28 deg.C for 15-20d, and adjusting pH to 3.5-3.8.

Preferably, the sterilized malus spectabilis ferment can be obtained by filling and sterilizing after the fermentation is finished or the live bacteria-containing malus spectabilis ferment can be obtained by aseptic filling.

The composite strain is Lactobacillus plantarum CGMCC3005, Lactobacillus bulgaricus CGMCC15159 and Streptococcus thermophilus CGMCC 15160.

Preferably, the ratio of the number of viable bacteria of the lactobacillus plantarum, the streptococcus thermophilus and the lactobacillus bulgaricus is 1-5:1-3: 1-3.

Preferably, the inoculation amount of the composite strain is 10⁵-10⁸cfu/mL。

The sugar may be a conventional sugar source such as white granulated sugar, glucose, fructose, and the like, preferably white granulated sugar.

The preparation method comprises the following steps:

1) squeezing fresh Chinese flowering crabapple fruit pulp and water in a ratio of 1:1 to obtain Chinese flowering crabapple raw juice, mixing the Chinese flowering crabapple raw juice and the water in a ratio of 1:2-5, and uniformly mixing the white granulated sugar with the addition amount of 3-10% of the total amount of the feed liquid;

2) mixing the activated composite strain with 10⁵-10⁸Inoculating cfu/mL, stirring, fermenting at 35-39 deg.C for 3-5 days, fermenting at 25-28 deg.C for 15-20 days, and adjusting pH to 3.5-3.8;

3) and after the fermentation is finished, filling and sterilizing to obtain sterilized malus spectabilis ferment or performing sterile filling to obtain live-bacteria-containing malus spectabilis ferment, and standing at room temperature for storage.

Preferably, the filling sterilization in step 3) adopts pasteurization: sterilizing at 75 deg.C for 15 min.

After the fermentation is finished, filling and sterilizing to obtain a sterilized Chinese flowering crabapple ferment finished product; and (3) obtaining a finished product of the crab apple enzyme containing the live bacteria through aseptic filling, wherein the aseptic filling is used for packaging the enzyme product in an aseptic environment instead of sterilizing the enzyme product, so that the enzyme product is ensured to be free of other mixed bacteria except the fermentation strain.

The pH value of the bactericidal and viable bacteria type product obtained by the method is 3.56 +/-0.4, and the total acid is 5.3 +/-0.3 g/100 g. The viable count of the viable type crab apple enzyme product is more than 10⁶cfu/mL。

Experiments in the step 1) prove that the Chinese flowering crabapple fruit pulp and water have a good juicing effect of 1:1, and the pulp is fine and smooth in the state, so that more juice can be squeezed, and the introduction of fruit residues is reduced.

Step 2) primary and secondary constant temperature stage fermentation aims at the stage proliferation of different zymophytes and the accumulation of metabolites thereof. The fermentation strains are directionally propagated and metabolized under different temperature conditions, the generated primary and secondary metabolites provide a foundation for the growth and reproduction of the strains in the subsequent stage, and simultaneously, the nutrient substances in the fermentation liquid can be better utilized and converted by the fermentation microorganisms.

And 3) filling and sterilizing by adopting pasteurization, wherein part of active substances are denatured and inactivated by common high-temperature sterilization to influence the nutrition and efficacy of the product, and the effective active ingredients of the fermented Chinese flowering crabapple enzyme can be retained to the maximum degree by the pasteurization treatment method.

The invention has the advantages and beneficial effects that:

(1) according to the invention, the lactobacillus plantarum CGMCC3005, the streptococcus thermophilus CGMCC15160 and the Lactobacillus bulgaricus CGMCC15159 are used, so that the sour and astringent taste of the crabapple is improved, the enzyme is finally bright orange yellow, the crabapple is rich in smell, the taste is slightly sour and free of impurities, and the additional value of the crabapple is improved.

(2) The invention adopts animal experiments: after 6-week-old male Kunming mice are selected and inoculated with malus gastrodiae enzyme for 28 days, compared with fecal microorganisms of blank mice, beneficial bacteria such as Lactobacillus and ruminococcus in intestinal tracts of the mice are increased, and harmful bacteria such as Citrobacter citrate are reduced. Thus, the crabapple ferment has the function of improving the intestinal flora microbial flora in the mouse.

(3) The invention adopts animal experiments: after the male Kunming mice with the age of 6 weeks are selected and filled with the begonia ferment for 28 days, the experimental group has better anti-depression capability than the blank group of mice in classical depression anxiety model experiments such as tail suspension experiment, forced swimming experiment, sugar water experiment and the like, which shows that the begonia ferment has the capability of relieving the depression of the mice.

Drawings

FIG. 1 volatile flavor chromatograms at 4d, 20d, 30d in the fermentor of example 1;

FIG. 2 is a graph showing the change in the content of dominant microorganisms in the fermentation process of example 1;

FIG. 3 is a graph comparing the activity time of the elevated plus maze laboratory mouse in the open arm region within 6 min;

FIG. 4 is a graph comparing the degree of sugar water preference of experimental mice.

Detailed Description

The present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention. Except as otherwise noted, the following examples were carried out using conventional techniques.

Example 1

1) Cleaning ripe and fresh undamaged Chinese flowering crabapple fruits, removing kernels and juicing. Uniformly mixing the Chinese flowering crabapple juice, the water material liquid and the white granulated sugar in a ratio of 1:4, wherein the addition amount of the white granulated sugar is 5%, and placing the mixture into a fermentation tank;

2) inoculating activated lactobacillus plantarum CGMCC3005, lactobacillus bulgaricus CGMCC15159 and streptococcus thermophilus CGMCC15160 into the Chinese flowering crabapple juice in the step 1), wherein the total inoculation amount is 10⁸cfu/mL, stirring uniformly, placing in an incubator, performing primary fermentation at 37 ℃ for 4d, and performing secondary fermentation at 28 ℃ for 31 d; the ratio of the viable count of the inoculated lactobacillus plantarum CGMCC3005, the inoculated lactobacillus bulgaricus CGMCC15159 and the inoculated streptococcus thermophilus CGMCC15160 is 2:1: 1;

3) after fermentation, sterile filling is carried out to obtain a finished product of the crab apple ferment, and the crab apple ferment is stored at 4 ℃;

the detection shows that the pH of the finished product of the malus spectabilis ferment is 3.58, the total acid is 5.3g/100g, and the viable count is more than 2 x 10⁶cfu/mL。

Example 2

1) Cleaning ripe and fresh undamaged Chinese flowering crabapple fruits, removing kernels and juicing, uniformly mixing Chinese flowering crabapple fruit juice with water-liquid ratio of 1:5 and the addition of 5% of white granulated sugar, and placing the mixture in a fermentation tank;

2) inoculating activated lactobacillus plantarum CGMCC3005, lactobacillus bulgaricus CGMCC15159 and streptococcus thermophilus CGMCC15160 into the Chinese flowering crabapple juice in the step 1), wherein the total inoculation amount is 10⁸cfu/mL, stirring uniformly, total inoculation amount is 10⁸cfu/mL, uniformly stirring, sealing, placing in an incubator, performing primary fermentation at 36 ℃ for 3d, performing secondary fermentation at 26 ℃ for 18d, and entering a post-maturation stage; the ratio of the viable count of the inoculated lactobacillus plantarum CGMCC3005, the inoculated lactobacillus bulgaricus CGMCC15159 and the inoculated streptococcus thermophilus CGMCC15160 is 1:1: 1;

3) after fermentation, sterile filling is carried out to obtain a finished product of the crab apple ferment, and the crab apple ferment is stored at 4 ℃;

the pH of the finished product of the malus spectabilis enzyme obtained in the embodiment is 3.54, the total acid is 5.1g/100g, and the viable count is more than 10⁷cfu/mL。

And filling and sterilizing the fermented ferment to obtain the sterilized begonia ferment, wherein the pH value is 3.54, and the total acid is 5.1g/100 g. Pasteurization is adopted for filling and sterilization: sterilizing at 75 deg.C for 15 min.

Example 3

1) Cleaning ripe and fresh undamaged Chinese flowering crabapple fruits, removing kernels and juicing. Uniformly mixing the Chinese flowering crabapple juice with the water-liquid material ratio of 1:3 and the addition amount of white granulated sugar of 4%, and placing the mixture in a fermentation tank;

2) inoculating activated lactobacillus plantarum CGMCC3005, lactobacillus bulgaricus CGMCC15159 and streptococcus thermophilus CGMCC15160 into the Chinese flowering crabapple juice in the step 1), wherein the total inoculation amount is 10⁸cfu/mL, stirring uniformly, total inoculation amount is 10⁸cfu/mL, stirring uniformly, performing primary fermentation at 37 ℃ for 5d and secondary fermentation at 28 ℃ in an incubator, and performing after-ripening after fermentation for 19 d; inoculated viable bacteria of lactobacillus plantarum CGMCC3005, lactobacillus bulgaricus CGMCC15159 and streptococcus thermophilus CGMCC15160The number ratio is 1:2: 2;

3) after fermentation, sterile filling is carried out to obtain a finished product of the crab apple ferment, and the crab apple ferment is stored at 4 ℃;

in this example, the pH of the finished product of the malus spectabilis enzyme is 3.55, the total acid is 5.5g/100g, and the viable count is greater than 3 × 10⁷cfu/mL。

And filling and sterilizing the fermented ferment to obtain the sterilized begonia ferment, wherein the pH value is 3.55, and the total acid is 5.5g/100 g. Pasteurization is adopted for filling and sterilization: sterilizing at 75 deg.C for 15 min.

Firstly, various indexes of the malus spectabilis enzyme fermentation process and the finished malus spectabilis enzyme product in the embodiment 1 are detected, and the obtained results are as follows:

(1) the change of the content (mg/L) of the organic acid before and after fermentation during the fermentation process is shown in Table 1:

TABLE 1

As can be seen from Table 1, the final lactic acid content was nearly 30 times that of the initial fermentation stage; the citric acid content is increased by 4 times; the malic acid content is reduced; organic acids such as succinic acid and fumaric acid are generated, and the succinic acid is known to be commonly used as a food sour agent for seasoning wine, candies and the like, and is naturally generated through fermentation without manual addition, so that the method is safer and more effective; the fumaric acid has strong buffering effect, can keep the pH of the solution at about 3.0, and has important effects on bacteriostasis and mildew resistance.

(2) The content of short chain fatty acid (ug/mL) in the crabapple enzyme fermentation is shown in Table 2:

TABLE 2

Time of fermentation	0d	4d	13d	20d	25d		31d
								Formic acid	1.11	7.01	3.26	3.73	3.31	2.98
Acetic acid	2.05	4.74	13.17	16.58	16.82	13.31
							Propionic acid	4.38	4.95	6.04	2.09	1.56	1.1
Butyric acid	0.34	0.48	0.78	1.18	0.69	0.15

When the time reaches 13d, the content of acetic acid reaches 13.17ug/mL, and the content of propionic acid reaches 6.04 ug/mL; the 4 short chain fatty acids have the most acetic acid content, followed by propionic acid. Short chain fatty acids, after being rapidly absorbed by the intestinal tract, both store energy and reduce osmotic pressure, and have important roles in maintaining the normal function of the large intestine and the morphology and function of colonic epithelial cells. Short chain fatty acids also promote sodium absorption, and butyric acid increases the yield of lactobacilli and reduces the number of escherichia coli.

(3) Volatile flavor change in fermenter

According to the change of the flavor substances analyzed by the graph 1, the 4 th d to the 31 th d of the alcohol substances are reduced to 5 from 8 to 9, the 4 th d to the 31 th d of the esters are reduced to 30 from 32 to 35, and the aldehyde, the acid and the ketone are basically unchanged in the fermentation process; during the fermentation process, the content of the ester component is the maximum, and the ratio is about 78.90%. According to the fermentation method, the strains can perform metabolic transformation on various flavor components of the crab apple juice under the synergistic effect, and finally the enzyme product is endowed with good taste.

(4) Change of dominant microorganism content in fermentation process

Microbial content in enzyme products

As can be seen from fig. 2 and table 3, the fermentation method of the present invention not only utilizes lactobacillus plantarum CGMCC3005, lactobacillus bulgaricus CGMCC15159, and streptococcus thermophilus CGMCC15160 to ferment the crabapple juice, but also actually utilizes yeast, acetic acid bacteria, and other lactic acid bacteria naturally existing in the crabapple juice (the lactic acid bacteria, acetic acid bacteria, and yeast mentioned in fig. 2 and table 3 all represent naturally existing strains and inoculated strains), and performs various metabolic transformations together. The saccharomycetes promote the utilization of sugar, generate ethanol through metabolism, provide a substrate for the fermentation of acetic acid bacteria, generate acetic acid (acetic acid) in products, enrich organic acids and other metabolites of the products, and further lay a material foundation for the exertion of the functions of the products.

Secondly, elevated plus maze and sugar water preference experiment is carried out by using the finished product of the malus spectabilis ferment in the embodiment 1

The experimental method comprises the following steps: the experimental mice were randomly divided into 2 groups of 8 mice each, blank and experimental groups, respectively, after one week of acclimation. The mice in the blank group and the experimental group are respectively and daily filled with 0.4mL of sterile normal saline and 0.4mL of crab apple enzyme. Gavage continued for 4 weeks. Behavioural experiments were performed at the end of the second week and at the end of the fourth week, respectively: elevated plus maze and sugar water preference experiments (specific experimental methods refer to HuiY, Dong-DongW, YueW, et al. Variantibrain-derivedNeurotrophic factor Val66 Methomorphism bacteria and parabiostatic peptides [ J ]. JNeeurosci, 2012, 32 (12): 4092-101), and mouse fecal microbiome detection.

(1) Elevated plus maze experiment:

the activity time of the mouse in the open arm region within 6min is counted, and the longer the residence time in the open arm region is, the better the anti-anxiety potential of the mouse is. In this experiment, it can be seen from FIG. 3 that the mice in the experimental group stay in the open arm region for a significantly longer time.

(2) Sweet water preference experiment

The mice are raised in cages (1 mouse/cage), 1 percent (g/mL) of sucrose water and pure water with the same volume are respectively added into each bottle (150 mL/bottle) for sugar water pre-adaptation training for 12 hours at 8:00 of the first morning; the first night is 8:00, the mouse drinking bottle and the sterile animal feed are all removed, and the mouse is deprived of food and water for 12 hours; the next morning, 8:00, sterile animal feed, and 1 bottle each of 1% sucrose water and 1% drinking water (150 mL/bottle) were placed in the squirrel cage, and 12 hours later, the volume of the remaining liquid was weighed, and the sugar water preference ratio of the mice was calculated. If the percentage of sugar water preference of the mice is significantly lower than that of the control group of mice, it is indicated that they exhibit depressive-like behavior.

According to the formula: sugar water preference/% -, A/B × 100

Wherein, A: consumption of syrup, mL; b: total liquid consumption, mL

In this experiment, it can be found from fig. 4 that the sugar water preference degree of the experimental group mice is close to 1 (100%), the sugar water preference degree of the blank group mice is about 0.4 (40%), and the sugar water preference degree of the experimental group is significantly higher than that of the control group.

Through the above high-elevated plus maze and sugar water preference classical experiment, the crabapple ferment has the potential of resisting depression.

(3) Mouse fecal microorganism detection results

The excrements of the mice in the experimental group have higher species abundance of microorganisms at the level of 2 families and genera than those in the blank group. At the family level, the experimental group mice feces after the stomach irrigation of the crabapple enzyme are increased with the families of erysipelothrix, lactobacillus and rhinitidae. The genus level increases Lactobacillus and Ruminococcus, and decreases Citrobacter. The lactobacillus is a common probiotic and has a promoting effect on the intestinal health of mice.

Claims (8)

1. The compound lactobacillus crabapple enzyme is characterized by comprising the following steps: squeezing Malus spectabilis fruits, adding sugar, performing primary fermentation at 35-39 deg.C for 3-5d, performing secondary fermentation at 25-28 deg.C for 15-20d, and finishing fermentation when pH reaches 3.5-3.8; the composite strain is Lactobacillus plantarum CGMCC3005, Lactobacillus bulgaricus CGMCC15159 and Streptococcus thermophilus CGMCC 15160.

2. The compound lactobacillus crabapple ferment of claim 1, wherein the ratio of the number of viable bacteria of lactobacillus plantarum, streptococcus thermophilus and lactobacillus bulgaricus is 1-5:1-3: 1-3.

3. The compound lactobacillus crabapple ferment of claim 1, wherein the compound lactobacillus crabapple ferment is obtained by filling and sterilizing after fermentation or is obtained by aseptic filling.

4. The compound lactobacillus crabapple enzyme according to claim 3, wherein the obtained sterilized crabapple enzyme and live-bacterium-containing crabapple enzyme product have a pH of 3.56 ± 0.4 and a total acid of 5.3 ± 0.3g/100 g; the viable count of the product containing viable bacteria malus spectabilis ferment is more than 10⁶cfu/mL。

5. The compound lactobacillus crabapple ferment of claim 1, wherein the inoculation amount of the compound strain is 10⁵-10⁸cfu/mL。

6. The compound lactobacillus crabapple ferment of claim 1, wherein the sugar is selected from white sugar, glucose, and fructose.

7. The preparation method of the compound lactobacillus crabapple ferment of claim 1, which is characterized by comprising the following steps:

1) squeezing fresh Chinese flowering crabapple fruit pulp and water in a ratio of 1:1 to obtain Chinese flowering crabapple raw juice, mixing the Chinese flowering crabapple raw juice and the water in a ratio of 1:2-5, and uniformly mixing the white granulated sugar with the addition amount of 3-10% of the total amount of the feed liquid;

2) mixing the activated composite strain with 10⁵-10⁸Inoculating cfu/mL, stirring, fermenting at 35-39 deg.C for 3-5 days, fermenting at 25-28 deg.C for 15-20 days, and adjusting pH to 3.5-3.8;

3) and after the fermentation is finished, filling and sterilizing to obtain sterilized malus spectabilis ferment or performing sterile filling to obtain live-bacteria-containing malus spectabilis ferment, and standing at room temperature for storage.

8. The method for preparing compound lactobacillus crabapple ferment of claim 7, wherein the filling sterilization in the step 3) is performed at 75 ℃ for 15min by pasteurization.

Images