Method for fermenting instant pearl barley with lactobacillus

Abstract

The application belongs to the technical field of food processing and particularly relates to a method for fermenting quick-cooking jobtea by lactobacillus; the method is used for treating jobteas by lactobacillus fermentation, and has a good improvement effect on steaming and cooking and nutritional quality of the jobteas. The application effectively reduces the hardness of the jobteas, improves the water absorption characteristics of the jobteas and improves the cooking performance on the premise of retaining the original appearance of the jobteas. Meanwhile, the lactobacillus fermentation can increase the contents of nutritional and functional components such as total starch, total protein, crude fat and gamma-aminobutyric acid of the jobteas, so that the jobteas have a light fragrance, and the nutritional quality and flavor of the jobteas are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food processing, and particularly relates to a method for fermenting quick-cooking jobtea by lactobacillus. BACKGROUND

[0002] Jobtea is a small grain with high nutritional and medicinal values, and has been favored by the market in recent years. However, the texture of jobtea is hard, and it is difficult to directly cook, which affects the consumer experience and limits the development of the industry. The current solution technology includes pre-soaking or pre-maturation through ultrasonic, microwave, high temperature and high pressure or puffing process, and then hot air drying to change the texture structure of jobtea. However, previous studies have shown that if the soaking time or temperature of the grain is insufficient, the grain is not easy to soften, and if the soaking time is too long or the temperature is too high, the grain is easy to reduce the taste and lose nutrients. The pre-maturation process such as ultrasonic, microwave, high temperature and high pressure or puffing is easy to cause cracks or deformation on the surface of jobtea, and high temperature and high pressure are also easy to destroy the nutritional components of the grain. In the hot air drying process, under the influence of high temperature and low humidity, the surface of jobtea is easy to crack and yellow.

[0003] In recent years, improving the physicochemical properties of cereal starch and cooking quality of food by fermentation process has become a research hotspot. Lactic acid bacteria is a kind of probiotics, including at least 18 genera and more than 200 species, such as Lactobacillus. Lactic acid bacteria fermentation can improve the texture, nutrition, flavor and shelf life of food, and has good economic benefits, can meet people's demand for green products and other advantages. The biochemical components of cereals are mainly starch, and some lactic acid bacteria strains (such as Lactobacillus paracasei, Lactobacillus amylovorus, Lactobacillus amylovorus and Lactobacillus plantarum, etc.) have strong amylase production capacity. Under appropriate fermentation conditions, through the joint action of amylase and lactic acid, the starch content or crystal structure of cereals can be changed, and then the physicochemical properties of starch and the quality of cereals can be changed. (Amadou et al., 2014) studied the fermentation of millet powder by Lactobacillus paracasei Fn023 to increase the starch content, and the starch crystal structure moderately lost integrity, and the starch paste texture became soft. (Zhang et al., 2012) showed that the crystal structure of rice starch changed after fermentation by Lactobacillus plantarum, and the functional properties of starch were fully improved. Under suitable fermentation conditions, fermented cereals can significantly improve the cooking quality, not only shorten the cooking time, but also improve the taste of cereals. (Cheng, 2018) studied the fermentation of brown rice by Lactobacillus plantarum JYI-3913, and the cooking time of the fermented brown rice was shortened by 20.17% compared with the raw material rice. After fermentation, the water absorption and volume expansion rate of brown rice increased, the hardness of brown rice decreased, the adhesion between rice grains was better, and the sensory quality was improved. (Chen et al., 2001) directly fermented early indica rice with 0.6% Rhizopus mould, 0.15% bread yeast and 0.15% alcohol yeast (inoculum 0.9%), and obtained early indica rice with soft taste and best sensory evaluation score. The nutritional quality of fermented cereals is also improved: (Yin et al., 2019) fermented coix seed powder with Lactobacillus plantarum NCU137, and the nutritional components of fermented coix seed powder, including free amino acids, free fatty acids, soluble dietary fiber and organic acids, were significantly higher than those of unfermented coix seed powder. The flavor and viscosity characteristics are stable, and the taste and appearance are more in line with the taste of consumers. (Wang et al., 2020) fermented germinated coix seed with Lactobacillus casei, and the nutritional components, including small molecule polypeptides, free amino acids and total phenol content, were significantly improved.

[0004] The existing lactic acid bacteria fermentation technology is mainly used for producing beverage or liquor, and mainly for fermenting the adlay powder. There is still lack of relevant technical report on how to directly ferment the adlay by using lactic acid bacteria to shorten the cooking time and improve the cooking quality and taste. The patent document with publication number CN107125569B discloses a method for improving the cooking performance of brown rice by fermentation of lactobacillus plantarum. The method is to mix the activated lactic acid bacteria with water and brown rice uniformly, then put them into a container with one-way air outlet, seal after filling or discharging the excess air; ferment and culture in a set temperature environment, take out after the fermentation is completed, dry and package, and then the brown rice product with easy cooking, good eating quality and high content of γ-aminobutyric acid (GABA) is obtained. Although there are relevant reports of other grains such as brown rice to provide theoretical reference, the biochemical components of adlay are unique, and the relevant fermentation technology of other grains cannot be directly applied to adlay. The development of instant adlay by using lactobacillus fermentation still needs to select suitable strains and explore suitable fermentation conditions according to the characteristics of adlay itself, so as to obtain instant adlay with low cost, easy cooking, good taste and high nutrition. SUMMARY

[0005] The present application provides a method for fermenting instant adlay by lactobacillus to solve the above problems.

[0006] Specifically, the method is implemented by the following technical solutions:

[0007] 1. A method for fermenting instant adlay by lactobacillus, which inoculates fermentation seed liquid on raw adlay, and performs vacuum freeze-drying after sealing fermentation; the fermentation seed liquid is obtained by culturing lactobacillus in MRS liquid medium for 24 hours.

[0008] Further, the lactobacillus is lactobacillus amylovorosus CGMCC1.3395.

[0009] Further, the specific method for fermenting instant adlay by lactobacillus is as follows:

[0010] (1) Preparation of fermentation seed liquid: pick single colony lactobacillus amylovorosus CGMCC1.3395 and inoculate it into MRS liquid medium, and culture it in a 37℃, 120r / min shaking bed for 24 hours until the final concentration of the bacterial liquid reaches 1-2×10 8 cfu / mL;

[0011] (2) Inoculation: after the raw adlay is removed and screened, it is loaded into a food-grade vacuum bag, and the fermentation seed liquid with a mass fraction of 0.50-1.00% of the raw adlay is inoculated thereon, then water is added, the water content is 30-40%, and the excess air is discharged and vacuum sealed.

[0012] (3) The raw adlay inoculated with the fermentation seed liquid in step (2) is placed in a 30-40℃ environment for fermentation for 8-16 hours, and the material is turned over every 6 hours during the fermentation process.

[0013] (4) Place the coix seed after fermentation in step (3) in a vacuum freeze dryer and dry for 5.5 to 7.5 hours until the moisture content of the coix seed is reduced to less than 10% (vacuum degree 0 to 5 Pa, dry at -55℃ for 30 minutes and then gradually increase the temperature to 30℃ for about 5 hours), and then pack it into bags.

[0014] I. Strain Screening

[0015] 1. Experimental Methods

[0016] 1.1 Experimental strains

[0017] Lactobacillus plantarum JYI-3913 (strain 1), Lactobacillus plantarum AS1.555 (strain 2), Lactobacillus amyloliquefaciens CGMCC1.3395 (strain 3), Lactobacillus amyloliquefaciens CGMCC1.3394 (strain 4), and Lactobacillus paracasei CGMCC1.12731 (strain 5). Strain 1 was ordered from Shandong Zhongke Jiayi Biotechnology Co., Ltd., strain 2 from Shanghai Preservation Biotechnology Center, and strains 3-5 from China General Microbiological Culture Collection Center.

[0018] 1.2 Activation and scale-up culture of strains

[0019] Activation of strains 1-3: Weigh 0.01 g (accurate to 0.01 g) of lyophilized bacterial powder and place it in a test tube containing 10 mL of sterile physiological saline (0.85% NaCl), homogenize for 1-2 min. Dilute the bacterial suspension 100 times, and spread 0.1 mL of the diluted solution onto MRS solid medium, and incubate anaerobically at 37°C for 48 h. Observe the colonies formed on different plates, pick a single colony and inoculate it into MRS liquid medium, and incubate anaerobically at 37°C for 48 h.

[0020] Activation of strains 4-5: Inoculate the bacterial suspensions onto MRS solid medium and incubate at 37°C for 3 generations (generally 24 hours). Pick a single colony from the MRS solid medium and inoculate it into MRS liquid medium, then incubate at 37°C for 24-36 hours.

[0021] Expanded culture: The tested strains were inoculated into MRS liquid medium at a volume fraction of 2% for expanded culture (37℃, 24–36 h) until the bacterial concentration reached 1–2 × 10⁻⁶. 8 cfu / mL.

[0022] 1.3 Morphological identification of strains

[0023] The strains 1-5 were inoculated into MRS liquid medium for 24 h, then streaked on MRS solid medium, and cultured anaerobically at 37℃ for 48 h. The colony growth characteristics, including colony size, shape, color, surface smoothness, edge roughness, and whether it was raised or not, were observed. The cultured strains were subjected to Gram staining, and the bacterial morphology was observed under a microscope and photographed. Red was Gram-negative bacteria, and purple was Gram-positive bacteria.

[0024] 1.4 Primary screening of amylase-producing ability of strains: starch ring color development method

[0025] The appropriate single colony on the plate was picked and inoculated into MRS liquid medium, and cultured anaerobically at 37℃ for 24 h. 5 μL of the bacterial solution was added to MRS-1% starch solid medium, and cultured anaerobically at 37℃ for 48 h. A certain amount of Lugol's iodine solution was added to the edge of the colony, and after sufficient penetration (about 20 min), the transparent ring color development around the colony was observed, and the colony diameter and transparent ring diameter were measured with an electronic digital caliper. The larger the transparent ring, the stronger the starch hydrolysis ability.

[0026] 1.5 Secondary screening of amylase-producing ability of strains: iodometric determination of starch degradation rate

[0027] The iodometric method was used to determine the starch degradation rate of the test strains.

[0028] Standard curve preparation: 100 μg / mL starch standard solution and 5 mmol / L iodine solution were prepared. According to Table 1, reagents were added to the test tubes and shaken uniformly. The absorbance was measured at 580 nm wavelength, and the starch-iodine color development reaction standard curve was drawn.

[0029] Table 1 Preparation of standard solutions of starch

[0030] Table1 Preparation of standard solutions of starch

[0031]

[0032] Starch degradation rate determination: the test strain was inoculated into MRS-1% starch liquid medium at an inoculation amount of 2%, and cultured anaerobically at 37℃ for 24 h. 3000 μL of the bacterial solution was centrifuged at 4500 r for 15 min. 400 μL of the supernatant was taken to measure the remaining starch content, 600 μL of PBS buffer solution was added, mixed, 2000 μL of iodine solution was added, shaken to develop color, and 2000 μL of the sample was transferred into a cuvette. The absorbance was measured at 580 nm wavelength, and the starch degradation rate was calculated. The starch content was calculated from the standard curve of starch-iodine color development reaction, and the starch degradation rate = (original starch content of the medium - remaining starch content of the medium) / original starch content of the medium × 100%.

[0033] 1.6 Enzyme activity determination: DNS method (dinitrosalicylic acid method)

[0034] The 3,5-dinitrosalicylic acid (DNS) method was used to determine the amylase activity of the test strain. Standard curve preparation: Prepare a 2 mg / mL maltose standard solution and add reagents to the test tubes according to Table 2, shake well, and place in a boiling water bath for 5 min, then cool with running water. Add distilled water to make up to 25 mL, use a No. 1 test tube as a blank control, and measure the absorbance at 540 nm, then draw the maltose standard curve.

[0035] Table 2 Preparation of standard solutions of maltose

[0036] Table2 Preparation of standard solutions of maltose

[0037]

[0038]

[0039] Enzyme activity determination: The test strain was inoculated into MRS liquid medium at a 2% inoculum and incubated, and enzyme activity was determined at 6, 12, 18, and 24 h, respectively. 4 mL of the liquid cultured bacteria were centrifuged (4°C, 4500 r, 15 min), 100 μL of the supernatant was taken, 400 μL of 1% starch PBS buffer solution was added and mixed well, and it was incubated in a 40°C water bath for 40 min. 1.5 mL of DNS reagent was added to terminate the reaction, and it was boiled in a water bath for 5 min, then cooled with running water. Deionized water was added to make up to 25 mL. The blank group took 1.5 mL of DNS reagent and added 0.5 mL of distilled water, then added distilled water to make up to 25 mL. The absorbance was measured at 540 nm, and the enzyme activity curve was drawn.

[0040] 1.7 Determination of growth curve of the strain

[0041] The test strain was inoculated into MRS liquid medium at a 1% inoculum and incubated at 37°C, 120 r / min on a shaker for 36 h. During the incubation process, 1 mL of culture was taken every 2 h, and 3 mL of sample was taken each time. The absorbance of each treatment was measured at 600 nm, with MRS liquid medium as the control.

[0042] 2 Results and analysis

[0043] 2.1 Morphological identification of the strain

[0044] Strains 1-5 were inoculated on MRS solid medium and incubated at 37°C anaerobically for 48h. All the tested strains showed round, milky white, opaque colony morphology, with smooth and sticky surface, regular edge, and no pigment production. Figure 1 a-e). Gram staining was performed on strains 1-5, and the cell morphology was observed under microscope using 100x oil immersion lens. All the tested strains were Gram-positive bacteria, with rod-shaped cell morphology, and single existence, about 2-4μm long. Figure 1 f-j). The colony and cell morphology of all the tested strains were consistent with the typical characteristics of Lactobacillus.

[0045] 2.2 Primary screening of amylase-producing ability of strains

[0046] The tested strains were cultured on starch chromogenic medium, and the starch hydrolysis ability of the tested strains was determined by judging the size of starch circle. The stronger the starch hydrolysis ability of Lactobacillus, the more obvious the color change of the starch degradation circle around the colony, and the culture medium became transparent with light blue color. The colony size and hydrolytic circle diameter of the strains after 48h of growth are shown in Table 3. It can be seen that after 48h of growth in the incubator at 37°C, the transparent circle of strains 3-4 was the largest and most obvious, followed by strains 1-2, and strain 5 had no starch circle. Further, the diameter of the starch degradation transparent circle of the tested strains was measured using an electronic digital caliper. The D / d value of strain 3 was the largest, followed by strain 4. It was preliminarily judged that the amylase activity of strains 3-4 was the highest (Table 3). Figure 2

[0047] Table 3 Colony size and hydrolytic circle diameter after 48h of strain growth

[0048] Colony size and hydrolytic circle diameter after 48h of strain growth

[0049]

[0050] Note: Different letters represent significant difference (p≤0.05), the same below.

[0051] 2.3 Secondary screening of amylase-producing ability of strains

[0052] According to the standard method, the standard curve of starch-iodine color reaction was drawn, and the linear regression equation was Y=0.0063X+0.0078, R 2 ​=0.9993, indicating good linearity. The tested strains were inoculated at a 2% inoculum into MRS-1% starch liquid medium and anaerobically cultured at 37℃ for 24 h. The starch degradation rate was then determined by iodometric titration. The results showed that strain 3 had the highest starch degradation rate (97.28%), exhibiting the strongest starch degradation ability and the best amylase production capacity; followed by strain 4 with a starch degradation rate of 30.44%; the remaining strains had starch degradation rates below 15% and poor amylase production capacity. Figure 3 ).

[0053] 2.4 Enzyme production capacity of the strain

[0054] The maltose standard curve equation for determining amylase activity using the DNS method is Y = 0.2625X - 0.0053, R 2 =0.9984, indicating good linearity. The changes in amylase activity during fermentation are as follows: Figure 4 As shown, the maximum enzyme activities of strains 1–5 were 20.23, 25.90, 36.94, 33.90, and 29.04 U / mL, respectively. Strain 3 exhibited the highest enzyme activity and strongest enzyme production capacity, followed by strain 4. Except for strain 1, the maximum enzyme production time for the other strains was 12 hours, after which the enzyme activity began to decline. This may be due to factors such as prolonged culture time, insufficient nutrient supply in the culture medium, and decreased pH inhibiting the activity of the strains.

[0055] 2.5 Growth capacity of the strain

[0056] The tested bacterial strains were inoculated at a 1% inoculum in MRS liquid medium and cultured at 37°C and 120 rpm in a constant temperature shaker for 36 h. Biomass was measured every 2 h during this period. Growth curves were plotted for the five strains. Figure 5 As shown: Strain 1 had a lag phase of 0–4 h, followed by logarithmic growth, reaching a stationary phase after 12 h, and then entering the death phase after 26 h. Strain 2 had a lag phase of 0–10 h, followed by logarithmic growth, reaching a stationary phase after 24 h, and then entering the death phase after 30 h. Strain 3 had a lag phase of 0–6 h, a logarithmic growth phase of 6–12 h, gradually stabilizing, and then entering the death phase after 30 h. Strain 4 experienced a 10 h lag phase followed by rapid growth, reaching a stationary phase after 12 h, and then slowly entering the death phase after 26 h. Strain 5 was similar to strain 1, with a lag phase of 0–4 h, a logarithmic growth phase of 4–16 h, and then entering the death phase after 26 h. The total biomass of the tested strains after entering the stationary phase was similar, but strain 4 was significantly lower than the other strains.

[0057] 3. Conclusion

[0058] Through morphological observation, starch transparent circle primary screening, iodine quantity method re-screening, starch degradation rate determination, enzyme activity curve and growth curve drawing, it is finally determined that the strain 3, i.e. Lactobacillus amylovorus CGMCC1.3395, has high efficient starch degradation capacity, with obvious starch degradation transparent circle, starch degradation rate as high as 97.28%, and the highest starch enzyme activity, and the strain culture has shorter lag phase, longer logarithmic phase and stationary phase, and high biological yield. The high starch content of grains is the most commonly used fermentation raw material, and Lactobacillus amylovorus has excellent amylase production capacity, which is expected to be applied to grain fermentation to improve fermentation effect, and has high use value and market prospect.

[0059] II. Safety evaluation of Lactobacillus amylovorus CGMCC1.3395

[0060] 1. Research content and result analysis

[0061] 1.1 Antibiotic sensitivity test

[0062] The antibiotic sensitivity test was performed according to the K-B drug sensitivity paper disc agar diffusion method, and the test results were determined according to the standard of Clinical and Laboratory Standards Institute (CLSI). If the colonies are sensitive to the antibiotic, a transparent circle will be formed around the drug sensitivity paper. The strain of Lactobacillus amylovorus CGMCC1.3395 was adjusted to a McFarland turbidity of 0.5 with 0.85% physiological saline, spread on MRS solid culture medium plates, placed with antibiotic drug sensitivity pieces, and incubated at 37°C for 24 hours in anaerobic conditions. The size of the transparent circle was recorded. The antibiotics used in the test, their contents and the size of the transparent circle are shown in Table 4.

[0063] In this study, 22 kinds of antibiotics were tested. The results showed that Lactobacillus amylovorus CGMCC1.3395 was sensitive to penicillin, ceftriaxone, cefotaxime, amikacin, gentamicin, streptomycin, tetracycline, chloramphenicol, lincomycin, clindamycin, erythromycin, teicoplanin, vancomycin, rifampicin, etc. It was sensitive to aminoglycosides and lipopeptides, and was resistant to ampicillin, kanamycin, ciprofloxacin, norfloxacin, ofloxacin, and levofloxacin.

[0064] Table 4 Results of Lactobacillus amylovorus CGMCC1.3395 drugsensitivity test(n=3)

[0065] Table 4 Results of Lactobacillus amylovorus CGMCC1.3395 drugsensitivity test(n=3)

[0066] Table 4 Results of Lactobacillus amylovorus CGMCC1.3395 drugsensitivity test(n=3)

[0067] Note: R-resistant, S-sensitive, I-intermediate sensitivity

[0068] 1.2 Drug resistance gene detection

[0069] According to the results of drug sensitivity experiment, the full sequence of common drug resistance genes of lactic acid bacteria and the genome sequence of Lactobacillus amylophilus CGMCC1.3395 were searched and downloaded from NCBI database (http: / / www.ncbi.nlm.nih.gov), and DNAman5.0 software (Lynnon Biosoft Company, USA) was used for alignment. Four pairs of primers were designed by selecting high homology fragments: kanamycin resistance gene aph, fluoroquinolone resistance gene qnr, β-lactam resistance gene bla-Ⅰ, bla-Ⅱ (corresponding to ampicillin). The designed primers were synthesized by Beijing Huada Gene Company, and the sequences and annealing temperatures are shown in Table 5.

[0070] Table 5 Primers used for PCR amplification of drug resistance genes

[0071] Table 5 Primers used for PCR amplification of drug resistance genes

[0072]

[0073] The methods of bacterial genomic DNA extraction, PCR amplification and electrophoresis were referred to Huang Xiaotang (2020). The experimental results showed that although Lactobacillus amylophilus CGMCC1.3395 showed resistance to ampicillin, kanamycin and fluoroquinolone antibiotics in drug sensitivity detection experiment, but the electrophoresis results of drug resistance gene detection showed that the above antibiotic related drug resistance genes were not detected in the bacteria. Figure 6

[0074] 1.3 Plasmid extraction

[0075] Refer to the instructions of Plasmid Extraction Kit (D1110, Solypus). The plasmid DNA of Lactobacillus amylophilus CGMCC1.3395 was detected by 1% agarose gel electrophoresis, and the strain containing plasmid (Lactococus lactis NZ3900, pBBR1MCS5-Tac-EGFP) was used as a control. The results are shown in Figure 7 The control strain appeared a fluorescent band in the lane, while the lane containing Lactobacillus amylophilus CGMCC1.3395 did not appear a fluorescent band, which indicated that the strain did not contain plasmid and had no risk as a storage host of drug resistance genes.

[0076] 1.4 Hemolysis phenomenon​

[0077] Hemolysis is one of the important indicators for in vitro safety testing of bacterial strains. Based on hemolytic characteristics, hemolysis can be divided into three categories: α-hemolysis, characterized by a greenish-yellow hemolytic zone around the colony, with poor pathogenicity to humans; β-hemolysis, characterized by a transparent hemolytic zone around the colony, with strong pathogenicity to humans; and γ-hemolysis, which is non-hemolytic and does not produce a hemolytic zone. Activated Lactobacillus amyloliquefaciens CGMCC1.3395 bacterial suspension was inoculated into blood agar plates (Haibo Biotechnology) by piercing 3-6 points with an inoculation needle, ensuring the bacteria were inoculated deep into the agar layer. The plates were then incubated upside down at 37°C for 24 hours, and the presence of hemolysis was observed. Figure 8 It is known that the bacterial colony has neither a grass-green hemolytic zone nor a transparent hemolytic zone, indicating that the bacterium does not have hemolytic properties.

[0078] 1.5 Nitroreductase Activity Assay

[0079] Lactobacillus amyloliquefaciens CGMCC1.3395 bacterial suspension was inoculated at a 1% inoculum into nitroreductase detection medium and incubated at 37℃ for 72 h. α-naphthylamine solution and p-aminobenzenesulfonic acid solution were added sequentially to the fermentation culture, and the color changes of the medium were observed. Escherichia coli DH5α was used as a positive control strain, and uninoculated nitroreductase detection medium served as a blank control (CK). Figure 9 It can be seen that the culture medium inoculated with Escherichia coli DH5α turned red, indicating a positive result; while the culture medium inoculated with CGMCC1.3395 strain did not change color, and its nitroreductase activity test result was negative, indicating that this strain cannot produce nitroreductase and will not reduce nitrates in the environment to nitrites, thereby producing harmful metabolites.

[0080] 1.6 Determination of azoreductase production capacity

[0081] The overnight activated strain CGMCC1.3395 was spread onto a substrate containing 5 mg / L of [agent / material]. -1 Place 7 μL of Direct Blue on solid culture medium and incubate at 37°C for 72 h. Figure 10 The absence of hydrolysis zones around the colonies indicates that this strain lacks the ability to produce azo reductase.

[0082] 1.7 Analysis of the ability of the strain to produce bioamines

[0083] Biogenic amines are alkaline, causing an increase in the pH of the culture medium and turning bromocresol purple in the medium purple. First, *Lactobacillus amyloliquefaciens* CGMCC1.3395 was activated overnight, and then transferred to cultures containing 1 g / L... -1 Histidine, 1 g / L -1The tyrosine was subcultured in a liquid medium, and subcultured every 24 h to induce the corresponding decarboxylase. After 10 times of subculture, the culture was coated on tyramine detection plate and histamine detection plate respectively, and the color change of the culture was observed after 72 h of culture. Figure 11 The Lactobacillus amylophilus CGMCC1.3395 showed yellow in both of the two media, indicating that the strain does not have the ability of amino acid decarboxylation, and will not produce harmful metabolites such as histamine and tyramine during growth.

[0084] 1.8 Mouse acute oral toxicity test

[0085] The mouse acute oral toxicity test of Lactobacillus amylophilus CGMCC1.3395 was completed by the Microorganism Analysis and Detection Center of Guangdong Provincial Institute of Microbiology. The test was implemented according to GB 15193.3-2014 Food Safety National Standard Acute Oral Toxicity Test. According to the classification standard of acute toxicity dose in the standard, the LD 50 >5000mg / kg.BW, belongs to the actual non-toxic level (Table 6).

[0086] Table 6 Results of acute oral toxicity test

[0087] Table 6 Results of acute oral toxicity test

[0088]

[0089] 2 Conclusion

[0090] Lactobacillus amylophilus is a probiotic bacteria with the functions of reducing cholesterol, stimulating intestinal enzyme activity, preventing and treating diarrhea, inhibiting bacteria and resisting viruses, etc. Lactobacillus amylophilus can be applied to yogurt, carbonated beverages and bread fermentation, etc. The Lactobacillus amylophilus CGMCC1.3395 with high amylase production screened in the present application has been applied to ferment rice to improve the quality of milled rice: the mechanical damage on the surface of starch granules of rice is reduced after fermentation (Shi Yue et al., 2020). In order to further verify the safety of the bacteria applied to food fermentation, a series of tests such as drug sensitivity test, drug resistance gene detection, plasmid detection, hemolysis test, nitroreductase activity, azoreductase production ability and biogenic amine production ability detection and mouse acute oral toxicity test are carried out, which prove that the strain has wide antibiotic sensitivity, does not contain common major drug resistance genes, and does not contain plasmids, so it has no risk of transferring drug resistance genes. The strain is negative in the detection of hemolysis test, nitroreductase activity, azoreductase production ability and biogenic amine production ability, indicating that it has no risk of producing harmful metabolites such as hemolysis, nitroreductase, azoreductase, histamine and tyramine. In summary, the strain is safe and harmless.

[0091] III. Process optimization of fermented Job's tears by Lactobacillus amylovorus CGMCC 1.3395

[0092] 1 Test design and method

[0093] 1.1 Single factor experiment

[0094] The effects of fermentation time (6, 12, 24, 48 h), fermentation temperature (20, 30, 40, 50 ℃), moisture content (30%, 40%, 50%, 60%), and inoculum size (0.1%, 0.5%, 1.0%, 1.5%) on fermented Job's tears were investigated. The pH of rice soup, optimal cooking time, broken rice rate, and rice puffing rate were used as detection indicators for single factor experiment. Raw rice (CK1) and Job's tears soaked at room temperature for 4 h (CK2) were used as controls. Fermented Job's tears were prepared as follows: 100 g (accurate to 0.1 g) of Job's tears after impurity removal were placed in a food-grade transparent plastic bag, inoculated with the strain and water, and stirred evenly. After removing the excess air, the bag was sealed and fermented in a shaker at the set temperature and time. The Job's tears were turned over every 6 h, and then dried in a vacuum freeze dryer for 6 h after fermentation.

[0095] 1.2 Orthogonal experiment

[0096] Based on the results of single factor experiment, the optimal cooking time, pH of rice soup, broken rice rate, and rice puffing rate were used as detection indicators for L 16 (5 4 ) orthogonal experiment to explore the optimal process parameters for fermenting Job's tears with Lactobacillus. The specific factor level settings are shown in Table 7, and the orthogonal experiment scheme is shown in Table 8.

[0097] Table 7 Factor level table of orthogonal array design

[0098] Table 7 Independent factors and levels of orthogonal array design

[0099]

[0100] 1.3 Cooking performance and nutritional quality analysis

[0101] Determination of the optimum steaming time of Coix seed: 50 mL distilled water was added into a 50 mL test tube, and 1 g of Coix seed was added after boiling. The time was immediately recorded. After boiling for a certain time, 10 Coix seeds were taken out, and the central part was observed to see if there was a white core. Then, the observation was carried out every 10 min, 5 min, and 1 min until the white core disappeared. The time was recorded, which was the optimum steaming time. After vacuum drying of Coix seed, the crack rate of milled rice was observed, and 100 Coix seeds were repeated in each group. After cooking, the rate of milled rice with a burst waist was counted, and 100 Coix seeds were repeated in each group. The above indexes were measured three times.

[0102] Water absorption rate of Coix seed during steaming: The raw material Coix seed W0 was accurately weighed and steamed under certain conditions. The mass of the cooked Coix seed W1 was weighed, and then the water absorption rate = (W1-W0) / W0x100%.

[0103] Volume expansion rate of Coix seed after cooking: A certain weight of raw material Coix seed was steamed (total mass 1 g) and placed in a 10 mL graduated cylinder. After 5 mL of water was added, the volume V0 was immediately measured. The total volume V2 of the cooked rice was V0-5. The total volume V1 of the raw material Coix seed was measured in the same way. The volume expansion rate = (V2-V1) / V1x100%.

[0104] The pH of the rice soup was measured by an electronic pH meter.

[0105] The hardness of milled rice and rice of Coix seed was measured by a digital grain hardness tester (WGW-1, Wenzhou Weidu). Each group was repeated for not less than 10 Coix seeds, and the unit was g.

[0106] Total starch content: 80% ethanol can separate soluble sugar and starch in the sample. Further, concentrated sulfuric acid is used to hydrolyze starch into glucose, and then anthrone colorimetry is used to determine the glucose content to calculate the starch content. This index uses this principle to determine the total starch content according to the plant starch content kit instruction manual of Suzhou Mengxi Biological Medicine Technology Co., Ltd.

[0107] Total protein content: The nitrogen content in the sample was determined by Kjeldahl method, and the result was multiplied by the conversion factor 6.25 to calculate the crude protein content. For details, refer to the national standard GB5009.5-2016, and the analysis instrument is Hainuo K9860 automatic Kjeldahl nitrogen determination instrument.

[0108] Crude fat content: Soxhlet extraction method was used to determine the fat content. The combined state of fat in food must be separated by strong acid, and the separated fat is easily dissolved in organic solvents. After hydrolysis of the sample by hydrochloric acid, the total content of free and combined fat is extracted by anhydrous ether or petroleum ether, and the solvent is removed. For details, refer to the national standard NY / T821-2019, and the analysis instrument is Hainuo SOX406 fat determination instrument.

[0109] γ-Aminobutyric acid (GABA): The absorption peak was determined according to the instructions of the γ-aminobutyric acid kit from Suzhou Mengxi Biomedical Technology Co., Ltd., and the peak value was 640 nm.

[0110] Total phenol content: determined according to the instructions of the plant total phenol reagent kit from Suzhou Mengxi Biomedical Technology Co., Ltd., with an absorption peak of 765 nm.

[0111] 2 Results and Analysis

[0112] This experiment aimed to select suitable fermentation conditions to reduce the cooking time of Job's tears without affecting the appearance and flavor of the polished rice and cooked rice. Therefore, four indicators were used as evaluation criteria: optimal cooking time, rice water pH, polished rice crack rate, and cooked rice breakage rate. Single-factor experiments were conducted on each indicator. Figure 12 ( ) and orthogonal experiments (Tables 8 and 9). Suitable fermentation conditions for quick-cooking Job's tears should meet the following aspects: (1) Short optimal cooking time. (2) Suitable pH of rice water. Previous studies have found that the taste threshold pH of human lactic acid is 4.50. Therefore, a good Job's tears fermentation process should meet the requirement that the pH of rice water is in the range of 4.5 to 7 (Piga et al. 2003), and the closer it is to neutral, the milder the sour taste brought by lactic acid produced by fermentation. (3) Low crack rate of polished rice. The surface of raw Job's tears often has slight cracks. This is mainly related to uneven water loss of Job's tears during processing and storage or damage from machine grinding. The influence of water during fermentation will deepen these cracks. If there are too many cracks or they are too heavy, it will affect its appearance and sales. (4) Low rate of rice bursting. When grains burst open when cooked into porridge, it means that the grains are softer and more easily absorb water and expand. After bursting, the nutrients are incorporated into the rice water, and the rice porridge will be thicker and more flavorful. However, rice that has cracked open has a reduced appearance of intact grains, and some consumers prefer to drink light rice porridge. Taking all these factors into consideration, we chose fermentation conditions that reduce the rate of rice cracking open.

[0113] 2.1 Single-factor experiment

[0114] Under conditions of 40% moisture content, 0.50% inoculum, and 40℃ fermentation temperature, both pH and optimal cooking time decreased with increasing fermentation time. Figure 12 (a, b) At a fermentation time of 48 hours, the optimal cooking time and lowest pH for Job's tears were 38.7 min and 3.97, respectively; these were 50 min and 2.57 lower than CK1. Although the cooking times for 48 hours and 24 hours of fermentation were short, the pH of the rice water was below 4.5. The crack rate of the polished rice was approximately 7% at 6 hours and 12 hours of fermentation, and the breakage rate of the cooked rice was 23.3% and 43.3%, respectively. Considering the optimal cooking time and pH, the optimal fermentation time was determined to be 12 hours.

[0115] Under the conditions of fermentation time 12 h, inoculation amount 0.50%, fermentation temperature 40℃, with the increase of moisture content, pH gradually increased (5.10 highest); the best cooking time was lower than 50 min, significantly lower than CK1 and CK2 but the variation range was not large among different moisture content treatments Figure 12 c,d) Considering that the broken rice rate and the rice belly-bursting rate were lower, the best moisture content treatment under this condition was 40%. Under this condition, the best cooking time was 46.7 min, the rice soup pH was 4.94, the broken rice rate was 4.7%, and the rice belly-bursting rate was 60%.

[0116] Under the conditions of fermentation time 12 h, moisture content 40%, fermentation temperature 40℃, with the increase of inoculation amount, the best cooking time and the rice soup pH gradually decreased Figure 12 e,f) When the inoculation amount was 0.10%, 0.50%, 1.00% and 1.50%, the best cooking time of Job's tears was 53.0, 46.7, 44.0, 43.0 min, and the rice soup pH was 5.43, 4.94, 4.83, 4.62, respectively. When the inoculation amount of bacterial liquid was 1.0% and 1.5%, the broken rice rate and the rice belly-bursting rate were significantly higher than those of 0.1% and 0.5%. Considering that the best cooking time of 0.5% bacterial liquid inoculation was significantly lower than that of 0.1%, the best inoculation amount was determined to be 0.50%.

[0117] Under the conditions of fermentation time 12 h, moisture content 40%, inoculation amount 0.50%, with the increase of fermentation temperature, the best cooking time first gradually decreased and then increased, and the pH first gradually decreased and then increased, both reached the minimum value at 40℃ Figure 12 g,h) When the fermentation temperature was 20, 30, 40, 50℃, the best cooking time was 50.3, 48.0, 46.7, 49.3 min, and the pH was 6.57, 5.50, 4.94, 6.62, respectively. Considering that the broken rice rate and the rice belly-bursting rate were lower at 30℃ than at 40℃, the best fermentation temperature was determined to be 30℃.

[0118] 2.2 Orthogonal test

[0119] From the orthogonal test results and range analysis (Table 8), the primary and secondary order of each factor was B > A > C > D for the best cooking time, A > D > B > C for the pH value, D > B > A > C for the broken rice rate, and A > B > C > D for the rice belly-bursting rate. The optimal scheme was A3B4C3D3 for the best cooking time, A1B2C1D1 for the pH value, A4B4C1D1 for the broken rice rate, and A1B1C1D2 for the rice belly-bursting rate.

[0120] Table 8. Orthogonal experimental design, results, and range analysis

[0121] Table 8 Orthogonal array design, results and range analysis

[0122]

[0123] Continued table

[0124]

[0125]

[0126] To further determine the influence of each factor on the four evaluation indicators, analysis of variance and significance tests were performed on the results in Table 8. The results of the analysis of variance are shown in Table 9. From the table, the F-test showed F(3, 35). 0.05 =2.87, F(3, 35) 0.01 =4.4, F(15, 32) 0.05 =1.98, F(15, 32) 0.01 =2.87. The effects of all factors on the optimal cooking time were highly significant (P≤0.001), with the order of importance being A>B>C>D. For the pH index, due to significant interactions among the factors (data not shown), only the inter-group error was recorded in the orthogonal experiment, and the differences between treatments were significant. Factors B and D had significant and highly significant effects on the crack rate of polished rice, respectively, while factors A and C had no significant effect on this index. Factors A and B had highly significant and significant effects on the breakage rate of cooked rice, respectively, while factors C and D had no significant effect on this index. Based on the results of range analysis and variance analysis, and considering the impact of balanced fermentation conditions on the four indicators, in order to achieve the best overall effect of short cooking time, moderate pH, low rice cracking rate and low rice breakage rate, the experimental combination was finally determined to be A2B2C2D2, namely, fermentation time of 12h, moisture content of 40%, bacterial inoculum amount of 0.5%, and fermentation temperature of 30℃. Under these conditions, the optimal cooking time, appearance and aroma of Job's tears fermented are more acceptable.

[0127] Table 9 L 16 (5 4 Orthogonal Experiment Variance Analysis Table

[0128] Table 9 L 16 (5 4 )Table of variance analysis of orthogonal test

[0129] (a) Optimal steaming time

[0130]

[0131] Note: R2= 0.911 (Adj R2= 0.880), *** means extremely significant difference, P < 0.001, 2

[0132] (b) pH

[0133]

[0134]

[0135] Note: R2= 0.999 (Adj R2= 0.999), ** means extremely significant difference, P < 0.01 2 2

[0136] (c) Milled rice crack rate

[0137]

[0138] Note: R2= 0.651 (Adj R2= 0.531), ** means extremely significant difference, P < 0.01; * means significant difference, P < 0.05. 2 2

[0139] (d) Rice broken rate

[0140]

[0141] Note: R2= 0.650 (Adj R2= 0.531), ** means extremely significant difference, P < 0.01; * means significant difference, P < 0.05. 2 2

[0142] 2.3 Changes in the cooking properties and nutritional components of Job's tears before and after fermentation

[0143] Compared with raw material rice (CK1) and conventional soaked rice (CK2), the color of milled rice of fermented Job's tears was slightly whiter, and the rice had a slight broken rate (P < 0.05) and a lower cooking loss (P < 0.01) after cooking. Figure 13 ​​​​​​​)。Further comparison of the cooking properties of fermented coix seed (A2B2C2D2) with CK1 and CK2 showed that the optimum cooking time of fermented coix seed was 48 min, which was 45.9% and 22.2% lower than CK1 and CK2, respectively. The water absorption and volume expansion of fermented coix seed were more than twice of CK1 and CK2. The hardness of milled rice and rice was much lower than that of raw material (CK1) and was about 1 / 2 of that of conventional soaked coix seed (CK2). Although the pH of coix seed soup was reduced due to lactic acid production, it was still higher than the sensitive pH threshold of 4.5 (Table 10). Overall, the texture of fermented coix seed was softer, more easily absorbed water and expanded, and thus was cooked faster.

[0144] Table 10 Changes of cooking properties of coix seed before and afterfermentation

[0145] Table 10 Changes of cooking properties of coix seed before and afterfermentation

[0146]

[0147] Compared with the control raw material (CK1) and the conventional soaked coix seed (CK2), the total starch, total protein and crude fat contents of fermented coix seed were increased, and the γ-aminobutyric acid content was increased to about 1.7 times of that of the raw material. The total phenol content was slightly lower than that of CK1 but was slightly higher than that of CK2.

[0148] Table 11 Changes of nutrients of coix seed before and afterfermentation

[0149] Table 11 Changes of nutrients of coix seed before and afterfermentation

[0150]

[0151] In summary, the beneficial effects of the present application are as follows:

[0152] The method of fermentation by Lactobacillus amylovorosus was used to treat the Job's tears, which had good improvement on the cooking and eating quality. The optimal fermentation conditions were determined as follows: fermentation time 12 h, fermentation temperature 30℃, moisture content 40%, inoculation amount 0.50%. The optimal treatment of fermentation Job's tears had the best cooking time shortened by 40.7 min, water absorption increased by 141.5%, and volume expansion rate increased by nearly 2.13 times. The hardness of milled rice and rice was reduced to less than 1 / 3 of the raw material, which effectively improved the cooking performance of Job's tears. At the same time, the fermentation of Job's tears by lactic acid bacteria also increased the nutritional components such as total starch, total protein, crude fat and functional components such as γ-aminobutyric acid, making Job's tears have a faint fragrance and improving the nutrition and flavor of Job's tears. BRIEF DESCRIPTION OF DRAWINGS

[0153] Figure 1 The colony and cell morphology of the test strain.

[0154] Figure 2 The starch transparent circle of the test strain.

[0155] Figure 3 The starch degradation amount of the test strain.

[0156] Figure 4 The enzyme production curve of the test strain.

[0157] Figure 5 The growth curve of the test strain.

[0158] Figure 6 The electrophoresis map of PCR amplification of drug resistance genes: lane 1 kanamycin aph, lane 2 fluoroquinolones qnr, lane 3-lactam class bla-Ⅰ, lane 4-lactam class bla-Ⅱ.

[0159] Figure 7 The plasmid extraction experiment results: lane 1 Lactococcus lactis NZ3900 (pBBR1MCS5-Tac-EGFP), lane 2 Lactobacillus amylovorosus CGMCC1.3395.

[0160] Figure 8 The hemolysis test results: a is the alpha hemolysis phenomenon of Streptococcus pneumoniae on blood agar, b is the beta hemolysis phenomenon of Bacillus cereus, and c is the gamma hemolysis phenomenon of Lactobacillus amylovorosus (CGMCC1.3395); a and b are used as positive controls and refer to the instruction manual of Haibo Biological Blood Agar Medium.

[0161] Figure 9 The nitroreductase activity detection results.

[0162] Figure 10 The azoreductase capacity determination results.

[0163] Figure 11 The detection result of biogenic amine produced by Lactobacillus plantarum CGMCC 1.3395.

[0164] Figure 12 The influence of fermentation conditions on the optimum cooking time of adlay, pH, milled rice crack rate and rice belly-bursting rate.

[0165] Figure 13 The physical comparison chart of milled rice and rice before and after adlay fermentation: raw material rice (CK1), conventional soaked adlay (CK2), optimum fermentation conditions (A2B2C2D2), milled rice (top) and rice (bottom). DETAILED DESCRIPTION

[0166] Example 1

[0167] 1. A method for fermenting adlay with Lactobacillus, specifically comprising the following steps:

[0168] (1) Preparing fermentation seed liquid: picking single colony of Lactobacillus plantarum and inoculating into MRS liquid medium, and culturing at 37℃, 120r / min for 24h;

[0169] (2) Inoculation: after removing impurities and screening, raw adlay is packed into food-grade vacuum bag, inoculating 0.50% fermentation seed liquid of raw adlay mass on it, then adding water with water content of 40%, discharging excess air, sealing, and fermenting;

[0170] (3) Placing raw adlay inoculated with fermentation seed liquid in step (2) into 35℃ condition for 12h fermentation, and turning over every 6h during the fermentation process;

[0171] (4) Placing fermented adlay in step (3) into vacuum freeze dryer for 6h drying, and packing.

[0172] Example 2

[0173] 1. A method for fermenting adlay with Lactobacillus, specifically comprising the following steps:

[0174] (1) Preparing fermentation seed liquid: picking single colony of Lactobacillus plantarum and inoculating into MRS liquid medium, and culturing at 37℃, 120r / min for 24h;

[0175] (2) Inoculation: after removing impurities and screening, raw adlay is packed into food-grade transparent plastic bag, inoculating 0.50% fermentation seed liquid of raw adlay mass on it, then adding water with water content of 35%, discharging excess air, sealing, and fermenting;

[0176] (3) Placing raw adlay inoculated with fermentation seed liquid in step (2) into 35℃ condition for 10h fermentation, and turning over every 6h during the fermentation process;

[0177] (4) The fermented Coix seed of step (3) is dried in a vacuum freeze dryer for 5.5 hours, and then is packed.

[0178] Example 3

[0179] 1. A method for fermenting quick-cooking Coix seed by Lactobacillus, comprising the following steps:

[0180] (1) Preparing fermentation seed liquid: picking single colony of Lactobacillus amylovorus and inoculating into MRS liquid medium, and culturing at 37°C and 120 r / min in a shaker for 24 hours;

[0181] (2) Inoculation: after the raw Coix seed is cleaned and sieved, it is put into a food-grade transparent plastic bag, and then 1.00% of the fermentation seed liquid of the raw Coix seed is inoculated thereon, water is added, the water content is 35%, and the excess air is discharged, and then the bag is sealed for fermentation;

[0182] (3) The raw Coix seed inoculated with the fermentation seed liquid of step (2) is fermented at 40°C for 15 hours, and the material is turned over every 6 hours during the fermentation;

[0183] (4) The fermented Coix seed of step (3) is dried in a vacuum freeze dryer for 7.5 hours, and then is packed.

[0184] The cooking properties of the fermented Coix seed prepared in the above examples and the raw Coix seed (CK1) are compared as follows:

[0185]

[0186] The nutritional components of the fermented Coix seed prepared in the above examples and the raw Coix seed (CK1) are compared as follows:

[0187]

[0188] The above results show that the cooking time is reduced in different degrees in Examples 1-3, the water absorption rate and the volume expansion rate of the rice grain are improved, the hardness of the milled rice and the rice is obviously reduced, the pH of the rice soup is within the sensitivity of human olfactory acid taste, the total starch, the total protein, the crude fat, and the active components are all increased, the γ-aminobutyric acid is significantly increased, and the total phenol content is slightly reduced but is similar to that of the raw Coix seed. It is shown that the cooking and nutritional quality of the fermented Coix seed by Lactobacillus amylovorus CGMCC1.3395 are improved.

[0189] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for rapidly cooking Job's tears through lactobacillus fermentation, characterized in that... Fermentation seed liquid was inoculated onto raw Job's tears, sealed for fermentation, and then vacuum freeze-dried; the fermentation seed liquid was obtained by inoculating Lactobacillus into MRS liquid culture medium and culturing for 24 hours. The lactobacillus mentioned is Lactobacillus amyloliquefaciens CGMCC1.3395; The aforementioned sealing fermentation specifically involves fermenting at 30–40°C for 8–16 hours with a moisture content of 30–40%, and turning the material over every 6 hours during the fermentation process.

2. The method for quickly cooking Job's tears by lactobacillus fermentation as described in claim 1, characterized in that, The fermentation seed liquid is prepared by: picking a single colony of Lactobacillus amyloliquefaciens and inoculating it into MRS liquid medium, then culturing it at 37℃ and 120 r / min in a shaker for 24 h, with a final bacterial concentration of 1–2 × 10⁻⁶. 8 cfu / mL.

3. The method for quickly cooking Job's tears by lactobacillus fermentation as described in claim 1, characterized in that, The inoculation amount of the seed fermentation liquid is 0.50 to 1.00% of the mass of the raw Job's tears.

4. The method for quickly cooking Job's tears by lactobacillus fermentation as described in claim 1, characterized in that, The aforementioned vacuum freeze-drying involves placing fermented Job's tears in a vacuum freeze dryer and drying them for 5.5 to 7.5 hours until the moisture content is reduced to less than 10%.

5. The method for quickly cooking Job's tears by lactobacillus fermentation as described in claim 4, characterized in that, The vacuum freeze drying process involves setting the vacuum level to 0–5 Pa, drying at -55°C for 30 minutes, and then gradually increasing the temperature to 30°C for 5 hours.

6. A method for rapidly cooking Job's tears by lactobacillus fermentation as described in any one of claims 1 to 5, characterized in that, The method is as follows: (1) Preparation of fermentation seed culture: A single colony of Lactobacillus amyloliquefaciens CGMCC 1.3395 was picked and inoculated into MRS liquid medium, and cultured at 37℃ and 120r / min in a shaker for 24h until the final bacterial concentration reached 1~2×10⁻⁶. 8 cfu / mL; (2) Inoculation: After the raw Job's tears are cleaned and screened, they are put into food-grade vacuum bags, and 0.50-1.00% of the raw Job's tears fermentation seed liquid is inoculated on them. Then water is added, with a water content of 30-40%. Excess air is removed and the bags are vacuum sealed for fermentation. (3) Place the raw material coix seed inoculated with the fermentation seed liquid in step (2) at 30-40℃ for 8-16 hours and turn it over every 6 hours during the fermentation process; (4) Vacuum freeze-dry the Job's tears prepared in step (3) for 5.5 to 7.5 hours until the moisture content of the Job's tears is less than 10%, and then pack them into bags.

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