A fermented enzyme of coix seed and a preparation method thereof

Abstract

The application discloses a kind of fermented enzyme of coix seed rice and a preparation method thereof, belong to the technical field of enzyme preparation, after being broken separately, coix seed rice is mostly irregular small block and small part of debris and broken grain after being broken, but after being broken together with a small amount of cereal, coix seed rice is almost no debris and broken grain, the integrity of coix seed rice after being broken is higher, it is shown that cereal has certain influence on the form of coix seed rice during the breaking process of coix seed rice, and the hull of cereal can also promote the fermentation effect in the subsequent fermentation process, the applicant compares and tests the amino butyric acid, triterpenes, phenols, flavonoids and free acid amount in the coix seed rice fermented enzyme prepared in the application and conventional coix seed rice enzyme, and finds that the value is increased by 1-3 times, such coix seed rice fermented enzyme is more beneficial to the elasticity of cardiovascular and reduction of blood fat, and is more beneficial to human health.

Description

A Job's tears fermentation enzyme and its preparation method Technical Field

[0001] This invention relates to the field of enzyme preparation and production, specifically to a coix seed fermentation enzyme and its preparation method. Background Technology

[0002] Job's tears, also known as coix seed, is a major high-quality coarse grain resource in Guizhou Province and other parts of my country. As a food and medicine homology food first released by the Chinese government, it has very high nutritional value. It is a nutritionally balanced grain with various properties such as anti-inflammatory, heat-clearing, water-draining, swelling-reducing, diuretic, spleen-strengthening, and lung-moistening effects.

[0003] Job's tears (coix seed) offer numerous health benefits, and it's commonly used in daily life to make health products and foods, such as Job's tears wine and Job's tears sesame paste. During an enzyme preparation experiment, the applicant wondered if using different microbial fermentation techniques in Job's tears fermentation might alter the final result of the fermented enzyme, and whether the fermented product would be better for the human body. Ultimately, after multiple experiments, the applicant adopted the Job's tears fermentation enzyme preparation method described in this application, resulting in a product that primarily uses a mixture of three different bacterial strains for co-fermentation, including Bacillus subtilis and Lactobacillus plantarum. The applicant tested the levels of aminobutyric acid, triterpenoids, phenols, flavonoids, and free acids in the prepared enzyme using *Limosilactobacillus reuteri*, and found that the values ​​were increased by 1-3 times. Such coix seed fermented enzyme is more beneficial to cardiovascular elasticity and lowering blood lipids. Summary of the Invention

[0004] The purpose of this invention is to overcome the aforementioned technical difficulties and provide a fermented coix seed enzyme that is more beneficial to cardiovascular elasticity and lowering blood lipids, as well as its preparation method.

[0005] To achieve the above objectives, the technical solution adopted is: a coix seed fermentation enzyme and its preparation method, comprising the following steps:

[0006] ① Raw material preparation: Select Job's tears that are free from rot, odor and pests, wash and filter them with clean water, then soak them in clean water for 6-12 hours, drain and set aside;

[0007] ② Crushing process: Add 1-4 parts of grains to the drained Job's tears from step ①, mix well, and then add them together to a crushing device to crush to 10-20 mesh for later use;

[0008] ③ Steaming: Steam the Job's tears (crushed together with the grains in step ②) for 30-60 minutes, then let it cool and set aside.

[0009] ④ Fermentation: Inoculate the cooled Job's tears from step ③ with Lactobacillus reuteri, Lactobacillus plantarum, and Bacillus subtilis, and then put them into a fermentation tank for fermentation for 48 hours;

[0010] ⑤ Stop fermentation: Heat the fermented Job's tears to 100℃ and steam for 10-30 minutes;

[0011] ⑥ Drying and grinding: Take out the cooked Job's tears from step ⑤ and place them in a drying oven to dry. Set the drying temperature of the drying oven to 40℃. Grind the dried Job's tears into baking powder using a grinding mill.

[0012] ⑦ Quality Inspection and Bottling: After sieving the baking powder, test it for coliform bacteria and pH before bottling.

[0013] Furthermore, the fermentation bacteria inoculated in step ④ include: Lactobacillus reuteri, Lactobacillus plantarum, Bacillus subtilis, and yeast.

[0014] Furthermore, in step ④, the *Lactobacillus reuteri*, *Lactobacillus plantarum*, *Bacillus subtilis*, and yeast are first subjected to a bacterial expansion treatment before inoculation.

[0015] Furthermore, in step four, the inoculation sequence of the microbial community is as follows: ① Lactobacillus plantarum ② Lactobacillus reuteri ③ Bacillus subtilis and yeast are inoculated simultaneously. Before inoculation, a compound enzyme is added to the fermentation tank and the Job's tears are stirred evenly. The compound enzyme includes pectinase, amylase, xylanase and cellulase.

[0016] Furthermore, in step ④, the ratio of Lactobacillus plantarum, Lactobacillus reuteri, Bacillus subtilis, and yeast is 1:0.8:1.5:0.1, and the coix seed is fermented in the fermentation tank for 48 hours.

[0017] Furthermore, the mass ratio of the grains added in step ② to the Job's tears is 1:5.

[0018] Furthermore, step ① includes: selecting Job's tears that are free from rot, odor, and pests, washing and filtering them with water twice, and then soaking them in water twice for 6-12 hours before use.

[0019] Furthermore, the grains used in step ② are fresh grains with their husks on, which have been washed, dried, and sterilized.

[0020] The beneficial effects of the above scheme are as follows: In the crushing process of this coix seed fermentation enzyme and its preparation method, when coix seeds are crushed alone, the crushed coix seeds are mostly irregular small pieces with a small amount of debris and particles. However, when a small amount of grains are added and crushed together with the coix seeds, the coix seeds are almost free of debris and particles, and the integrity of the crushed coix seeds is higher. This indicates that the grains have a certain influence on the morphology of the coix seeds during the crushing process, and the husks of the grains can also promote the fermentation effect in the subsequent fermentation process. The applicant compared the content of aminobutyric acid, triterpenoids, phenols, flavonoids and free acids in the coix seed fermentation enzyme prepared in this application and conventional coix seed enzyme, and found that the values ​​were 1-3 times higher. Such coix seed fermentation enzyme is more beneficial to cardiovascular elasticity and lowering blood lipids, and is more beneficial to human health. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] Example 1

[0023] A coix seed fermentation enzyme and its preparation method, comprising the following steps:

[0024] ① Raw material preparation: Select Job's tears that are free from rot, odor and pests, wash and filter them with clean water, then soak them in clean water for 6-12 hours, drain and set aside;

[0025] ② Crushing process: Add grains to the drained Job's tears from step ① and mix well. Then add them together to a crushing device and crush to 10-20 mesh for later use. The mass ratio of the added grains to the Job's tears is 1:5.

[0026] ③ Steaming: Steam the Job's tears (crushed together with the grains in step ②) for 30-60 minutes, then let it cool and set aside.

[0027] ④ Fermentation: After cooling in step ③, the Job's tears are inoculated with Lactobacillus reuteri, Lactobacillus plantarum and Bacillus subtilis, and then put into a fermentation tank for fermentation for 48 hours. The ratio of Bacillus subtilis, Lactobacillus plantarum and Lactobacillus reuteri is 1:0.2:1.5.

[0028] ⑤ Stop fermentation: Heat the fermented Job's tears to 100℃ and steam for 10-30 minutes;

[0029] ⑥ Drying and grinding: Take out the cooked Job's tears from step ⑤ and place them in a drying oven to dry. Set the drying temperature of the drying oven to 40℃. Grind the dried Job's tears into baking powder using a grinding mill.

[0030] ⑦ Quality Inspection and Bottling: After sieving the baking powder, test it for coliform bacteria and pH before bottling.

[0031] The crushing device can be a crusher as used in existing technologies.

[0032] In this method of preparing coix seed fermentation enzyme, when coix seeds are crushed alone, they mostly appear as irregular small pieces with a small amount of debris and fragments. However, when a small amount of grains are added and crushed together with the coix seeds, the resulting coix seeds appear to have almost no debris or fragments, and the integrity of the crushed coix seeds is higher. This indicates that the grains have a certain influence on the morphology of the coix seeds during the crushing process, and the husks of the grains can also promote the fermentation effect in the subsequent fermentation process. The applicant compared the levels of aminobutyric acid, triterpenoids, phenols, flavonoids, and free acids in the coix seed fermentation enzyme prepared in this application with those in conventional coix seed enzymes, and found that the values ​​were 1-3 times higher. Such coix seed fermentation enzyme is more beneficial to cardiovascular elasticity and lowering blood lipids, and is more beneficial to human health.

[0033] Example 2

[0034] Based on Example 1, in order to prove whether the addition of grains for co-crushing in step ② was accidental, the applicant chose to add hulled grains and millet in step ② to conduct the following control experiment.

[0035] Control group ①: Job's tears were crushed separately in step ②;

[0036] Control group ②: In step ②, hulled grains were added to Job's tears and mixed well, and then the mixture was added to a crushing device for crushing.

[0037] Control group ③: In step ②, millet was added to Job's tears and mixed well, and then the mixture was added to a crushing device for crushing.

[0038] Control group ④: In step ②, add grains to the Job's tears and mix well, then add them together to the crushing device for crushing.

[0039] Control group ⑤: In step ②, red beans were added to Job's tears and mixed well, and then the mixture was added to a crushing device for crushing.

[0040] In the control group mentioned above, the quality of Job's tears was consistent. The added millet, hulled grains, rice grains, and red beans were in equal amounts. The remaining steps were consistent with the preparation method in Example 1. The morphology of the crushed Job's tears, the fermentation effect, and the levels of GABA, triterpenoids, phenols, flavonoids, and free acids were tested. The results are as follows:

[0041] The form of Job's tears:

[0042] In control group ①, the broken Job's tears were in the form of both large and small pieces, with a lot of fragments and particles;

[0043] In control group ②, the Job's tears showed some fragmentation, and some grains were directly broken into fragments;

[0044] The crushing effect of control group ③ was similar to that of control group ①, but the millet was either crushed into fragments or not crushed at all.

[0045] In control group ④, the Job's tears were broken into irregular lumps with almost no fragments or particles;

[0046] In control group ⑤, most of the red beans were not broken, and some of the Job's tears were broken, with only the outer skin damaged, indicating poor breaking effect.

[0047] After preparing the control groups ①-⑤ according to the preparation method of Example 1, the applicant tested the fermentation effect of coix seed and the amount of aminobutyric acid, triterpenoids, phenols, flavonoids and free acids in the control groups ①-⑤. It was found that the fermentation time of control group ① was the longest, the fermentation time of control groups ②, ③ and ⑤ were similar, and the fermentation effect was better than that of control group ①. Among them, the fermentation time of control group ④ was the shortest and the fermentation effect was the best. The amount of aminobutyric acid, triterpenoids, phenols, flavonoids and free acids in the control groups was 3 times that of control group ①, nearly 2.5 times that of control groups ③ and ⑤, and nearly twice that of control group ②.

[0048] The applicant found that in step ②, the added grains were fresh grains, which not only had the same effect on the crushing of Job's tears in step ② as control group ④, but also had a better subsequent fermentation effect on Job's tears and a higher number of beneficial molecules in subsequent tests.

[0049] The applicant believes that adding hulled grains during the crushing process may have influenced the crushing process of the Job's tears due to the texture of the hulled grains, resulting in fewer broken pieces and debris. Furthermore, the outer shell of the grains may improve the fermentation process. The optimal ratio of grains to Job's tears in step ② is 1:5, avoiding both excessive grains affecting crushing and fermentation and insufficient grains leading to debris and poor fermentation.

[0050] Example 3

[0051] Based on Examples 1 and 2, the applicant conducted the following comparative experiment in step ④ to investigate whether different strains of bacteria selected during inoculation affected the content of beneficial components in coix seed enzymes. Multiple comparative experiments were conducted using various different strains of bacteria.

[0052] Before inoculating Job's tears with Lactobacillus reuteri, Lactobacillus plantarum, and Bacillus subtilis, the applicant first fermented Job's tears with yeast powder or natural fermentation. After multiple experiments, the applicant found that only the enzyme prepared by using Lactobacillus reuteri, Lactobacillus plantarum, and Bacillus subtilis had the highest content of GABA, triterpenoids, phenols, flavonoids, and free acids, and had the best effect.

[0053] Fermented *Lactobacillus plantarum* has certain effects on human immune regulation, inhibits pathogenic bacteria, lowers serum cholesterol levels, prevents cardiovascular disease, maintains intestinal flora balance, promotes nutrient absorption, alleviates lactose intolerance, and inhibits tumor cell formation, among other beneficial effects. Fermented *Lactobacillus reuteri* has effects such as reducing inflammation and strengthening immunity, maintaining intestinal flora harmony, and lowering cholesterol. Fermented *Bacillus subtilis* inhibits pathogenic bacteria, promotes the growth of beneficial anaerobic bacteria, produces lactic acid and other organic acids, lowers intestinal pH, and indirectly inhibits the growth of other pathogenic bacteria. It increases immunoglobulin and antibody levels, enhances cellular and humoral immunity, and improves herd immunity. It synthesizes enzymes such as α-amylase, protease, lipase, and cellulase, which work in the digestive tract alongside digestive enzymes in animals (humans).

[0054] After selecting Bacillus subtilis, Lactobacillus plantarum, and Lactobacillus reuteri as the inoculation strains for step ④, the applicant made several adjustments to the inoculation order of the three strains and conducted multiple comparative experiments. The final experimental results showed that only when the inoculation order was Lactobacillus plantarum, Lactobacillus reuteri, and Bacillus subtilis, the levels of phenols and flavonoids in the final coix seed enzyme were 2-3 times higher than those of other inoculation orders. Therefore, the applicant believes that the order of inoculation of the microbial community in step ④ directly affects the fermentation effect of coix seed enzyme, and the effect is best when the inoculation order is Lactobacillus plantarum, Lactobacillus reuteri, and Bacillus subtilis.

[0055] After confirming the inoculation order of the three strains, the applicant accidentally mixed a small amount of yeast into the inoculation of Bacillus subtilis while inoculating Job's tears. As a result, the applicant found that the fermented Job's tears with the added yeast had a stronger aroma, looked softer, and had a richer taste. The amount of cellulose, free acid, and phenol in the fermented Job's tears increased by 1.2%-3%, resulting in better health benefits.

[0056] Therefore, the applicant conducted multiple batches of inoculation experiments on the same Job's tears using the same bacterial strain, and then collected data on the prepared enzyme. The inoculation comparison experiments included: ① *Lactobacillus plantarum* mixed with yeast, *Lactobacillus reuteri*, and *Bacillus subtilis*; ② *Lactobacillus plantarum*, *Lactobacillus reuteri* mixed with yeast and *Bacillus subtilis*; ③ *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Bacillus subtilis*, and yeast. The results showed that none of the experiments were as good as the experimental data obtained by chance from the mixture of *Lactobacillus plantarum*, *Lactobacillus reuteri*, and *Bacillus subtilis* with yeast. Therefore, the applicant believes that the fermentation of yeast mixed with *Bacillus subtilis* may have created a fermentation environment beneficial to Job's tears, and also improved the survival environment of the previously inoculated *Lactobacillus plantarum* and *Lactobacillus reuteri*, thus increasing the number of beneficial molecules obtained subsequently.

[0057] The applicant believes that during the fermentation process, it is highly likely that when *Lactobacillus plantarum* was first inoculated, the lactobacillus fermented the Job's tears and grains, converting most of the sugars into lactic acid, while also producing a certain amount of antimicrobial peptides, extracellular polysaccharides, and various metabolites. *Lactobacillus reuteri* was able to produce glucan and fructan during fermentation. The two bacterial groups fermented one after the other, improving and preparing the fermentation environment for the subsequent inoculation of *Bacillus subtilis*. When yeast was added at the same time as *Bacillus subtilis*, the yeast created a fermentation environment beneficial to the Job's tears and also improved the survival environment of the previously inoculated *Lactobacillus plantarum* and *Lactobacillus reuteri*. Overall, the fermentation effect on the three main bacterial groups was optimal.

[0058] In the inoculation experiment, the applicant conducted multiple comparative experiments on the inoculation amount of the three types of bacteria. It was found that the ratio of Lactobacillus plantarum, Lactobacillus reuteri, Bacillus subtilis and yeast added in ④ was 1:0.8:1.5:0.1, and the fermentation time was 48h for the best effect.

[0059] The applicant performed a bacterial expansion treatment on the three bacterial groups to be inoculated before vaccination. This bacterial expansion treatment can greatly reduce the cost of using the bacterial groups and shorten the usage cycle.

[0060] To ensure the nutritional content of the Job's tears before inoculation, the applicant added a complex enzyme to the fermentation tank and stirred the Job's tears evenly before inoculation. The complex enzyme included pectinase, amylase, xylanase and cellulase.

[0061] Example 4

[0062] The applicant, having gained some experience and inspiration from their usual preparation of various enzyme fermentations, added a compound enzyme and fresh Gastrodia elata to the fermentation tank before inoculation, stirring thoroughly before proceeding with fermentation. The applicant found that the phenolic content of the fermented enzyme prepared using this method was 1.2 times that of the enzyme in Example 3, while the organic acid content increased to 1.4 times. Furthermore, the enzyme product prepared using this method was more beneficial in improving vascular elasticity and also possessed various effects such as lowering blood pressure, resisting myocardial ischemia, resisting arrhythmia, anti-inflammation, anti-aging, anti-oxidation, anti-radiation, vasodilating, anticoagulation, and antithrombosis. The applicant found that the enzyme produced during fermentation had the best effect when the mass ratio of fresh Gastrodia elata to Job's tears was 1:10.

[0063] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0064] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing coix seed fermentation enzyme, characterized in that: The process includes the following steps: ① Raw material preparation: Select Job's tears free from spoilage, odor, and pests, wash and filter with clean water, then soak in clean water for 6-12 hours, drain and set aside; ② Crushing: Add 1-4 parts of fresh grains with husks to the drained Job's tears from step ①, mix well, and then crush together to 10-20 mesh using a crushing device. The mass ratio of grains to Job's tears is 1:5; ③ Steaming: Steam the crushed grains and Job's tears from step ② together for 30-60 minutes, then let cool and set aside; ④ Fermentation: Inoculate the cooled grains and Job's tears from step ③ sequentially with Lactobacillus plantarum, Lactobacillus reuteri, Bacillus subtilis, and yeast, and ferment in a fermentation tank for 24-48 hours; ⑤ Termination of fermentation: Heat the fermented grains and Job's tears to 100℃ and steam for 10-30 minutes; ⑥ Drying and grinding: Take out the cooked grains and Job's tears from step ⑤ and place them in a drying oven to dry. The drying temperature of the drying oven is set to 40℃. The dried grains and Job's tears are then ground into baking powder using a grinding mill. ⑦ Quality inspection and bottling: After sieving the baking powder, test it for coliform bacteria and pH before bottling. The ratio of Lactobacillus plantarum, Lactobacillus reuteri, Bacillus subtilis and yeast added in step ④ is 1:0.8:1.5:0.

1.

2. The method for preparing coix seed fermentation enzyme according to claim 1, characterized in that: The fresh grains with husks in step ② are fresh grains with husks that have been washed, dried, and sterilized.