A yellow pulp water fermentation preparation and its application in the root growth and bacterium inhibition of bean sprouts

Abstract

The application discloses the technical field of microbial preparation, and relates to a yellow slurry water fermentation preparation and application of the yellow slurry water fermentation preparation as a bean sprout rooting bacteriostatic agent. The yellow slurry water fermentation preparation is obtained by mixed fermentation of Kluyveromyces marxianus NNB-2 and Lactobacillus paracasei LMX1 on a fermentation substrate containing yellow slurry water, and the yellow slurry water fermentation preparation is further applied as a rooting bacteriostatic agent in bean sprout culture. Subsequent test research shows that application of the yellow slurry water fermentation preparation in the process of bean sprout culture can significantly promote growth indexes such as the rooting and germination rate of bean sprouts, and can further improve the disease resistance and rot resistance of bean sprouts. Meanwhile, the yellow slurry water fermentation preparation has a significant advantage in reducing the content of harmful substances such as sulfite, nitrite and lead in bean sprouts, and improving the concentration of ascorbic acid and the activity of SOD enzyme, thereby providing a scientific basis for the production of green, high-quality and functional sprout vegetables.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial preparation, in particular to a yellow slurry fermentation preparation and its application in the root growth and bacteriostatic agent of bean sprouts. BACKGROUND

[0002] A large amount of wastewater is produced in the production process of bean products such as tofu and dried bean curd. Since the wastewater contains color substances such as flavonoids and carotenoids, and is a yellow mixture liquid, the soybean processing wastewater is also called "yellow slurry water". Studies have shown that the soluble components in soybeans are lost with the yellow slurry water during the production process of bean products. Therefore, the yellow slurry water contains soybean protein, polysaccharide, fat, pigment and mineral matter, and also contains bioactive substances such as soybean isoflavones, soybean oligosaccharides and soybean saponins, which are rich in nutrients. At present, the resource utilization of yellow slurry water is mainly to recover and utilize its functional components, or to develop processed foods or to develop feed by microbial fermentation. Commonly used microorganisms include yeast, lactic acid bacteria and Bacillus.

[0003] Bean sprouts are rich in protein, amino acids, vitamins and minerals. They are not only crisp and delicious, but also have the health care effects of clearing heat and improving eyesight, tonifying qi and nourishing blood, eliminating dampness and swelling, and beautifying and moisturizing the skin. At present, the production process of bean sprouts is very mature. In recent years, the research trend of bean sprout production is to improve the yield and quality of bean sprouts by adding nutrients, so as to improve their nutritional value and economic value. For example, organic fertilizer, plant enzymes and other methods are added. In addition, during the cultivation of bean sprouts, diseases such as root rot and mold spots often occur, which will have a great impact on the yield and quality of bean sprouts. Therefore, during the cultivation of bean sprouts, attention should also be paid to the prevention and control of bean sprout diseases. Bean sprout disinfectants are often used for treatment, which will cause the quality of bean sprouts to decline. Some unscrupulous people abuse hormones harmful to human body for the sake of profit, and breed bean sprouts quickly, which leads to the "toxic bean sprout incident" and poses a serious threat to public health. Related harmful hormones have been banned for use in soybean sprout cultivation. Developing new chemical hormone substitutes to enable soybean sprouts to grow quickly under safe conditions has become a research hotspot.

[0004] In summary, the present application develops a yellow slurry fermentation agent and applies it to the root growth and bacteriostatic agent of bean sprouts, aiming to promote the growth and development of bean sprouts, resist disease and corruption, reduce harmful substance residues, and promote the improvement of nutritional indicators, which is of great significance for the resource utilization of yellow slurry water and the production of green, safe and healthy sprout vegetables. SUMMARY

[0005] The present application aims to overcome the shortcomings of the prior art and provides a yellow slurry fermentation preparation and its application in the root growth and bacteriostatic agent of bean sprouts.

[0006] The present application achieves the above-mentioned purpose by the following technical solutions:

[0007] As a first aspect of the present application, a yellow serum water fermentation preparation is provided, which is obtained by inoculating mixed bacteria of Kluyveromyces marxianus NNB-2 and Lactobacillus paracasei LMX1 into a fermentation substrate containing soybean yellow serum water and then performing liquid fermentation.

[0008] As a further optimization scheme of the present application, the Lactobacillus paracasei LMX1 is preserved in the China Center for Type Culture Collection, with a strain preservation number of CCTCC NO: M20211073 and a preservation time of August 24, 2021.

[0009] The Kluyveromyces marxianus NNB-2 is preserved in the China Center for Type Culture Collection, with a strain preservation number of CCTCC NO: M20221265 and a preservation time of August 9, 2022.

[0010] As a further optimization scheme of the present application, the fermentation substrate further includes wort in addition to the soybean yellow serum water, and the volume ratio of the soybean yellow serum water to the wort is 4:1.

[0011] As a further optimization scheme of the present application, the volume ratio of the Kluyveromyces marxianus NNB-2 to the Lactobacillus paracasei LMX1 is 1:1.

[0012] As a further optimization scheme of the present application, the preparation method of the yellow serum water fermentation preparation includes the following steps:

[0013] (1) The Kluyveromyces marxianus NNB-2 and the Lactobacillus paracasei LMX1 are respectively activated, recovered and liquid cultured to obtain seed liquids;

[0014] (2) The seed liquids of the Kluyveromyces marxianus NNB-2 and the Lactobacillus paracasei LMX1 are mixed, inoculated into the fermentation substrate at 10% of the total volume of the fermentation substrate, and uniformly mixed, and then liquid fermentation is performed at a fermentation temperature of 30°C for 72 hours, after which the supernatant is obtained by filtration.

[0015] As a second aspect of the present application, a bean sprout rooting bacteriostatic agent is provided, which is obtained by diluting the above-mentioned yellow serum water fermentation preparation with 300-500 times water.

[0016] As a third aspect of the present application, the use of the above-mentioned bean sprout rooting bacteriostatic agent in improving bean sprout growth indicators is provided, characterized in that the growth indicators include at least one of rooting and germination rate, stem length, fresh weight or water content.

[0017] As a fourth aspect of the present application, there is provided an application of the bean sprout rooting bacteriostatic agent as described above in promoting the disease resistance and decay resistance of the bean sprouts.

[0018] As a fifth aspect of the present application, there is provided an application of the bean sprout rooting bacteriostatic agent as described above in reducing the content of harmful substances in the bean sprouts, the harmful substances including at least one of sulfite, nitrite or lead element.

[0019] The present application has the following beneficial effects:

[0020] The present application utilizes Kluyveromyces marxianus NNB-2 and Lactobacillus paracasei LMX1 to ferment a fermentation substrate containing yellow serum water to obtain a yellow serum water fermentation preparation, and further applies the yellow serum water fermentation preparation as a rooting bacteriostatic agent to bean sprout cultivation. Through subsequent experimental research, it is found that the application of the yellow serum water fermentation preparation during the cultivation of bean sprouts can significantly promote the rooting and germination rate of bean sprouts and other growth indicators, and can further improve the disease resistance and decay resistance of bean sprouts, while reducing the content of harmful substances such as sulfite, nitrite and lead in bean sprouts, and improving the concentration of ascorbic acid and the activity of SOD enzyme. The present application provides a scientific basis for the production of green and high-quality functional sprout vegetables. DETAILED DESCRIPTION

[0021] The following further detailed description of the present application is necessary to point out that the following detailed description is only used to further illustrate the present application, and cannot be understood as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application according to the above application content.

[0022] Reagents and materials

[0023] The reagents and materials used below are commercially available products unless otherwise specified.

[0024] Strain No. 1 is Lactobacillus paracasei LMX1, which is screened by the Institute of Agricultural Products Processing of Anhui Academy of Agricultural Sciences and preserved by the China Center for Type Culture Collection, with the strain preservation number CCTCC NO: M20211073 and the preservation time August 24, 2021.

[0025] The strain No. 2 is Kluyveromyces marxianus NNB-2, which is screened by the Institute of Agricultural Products Processing of Anhui Academy of Agricultural Sciences and is preserved in China Center for Type Culture Collection (CCTCC) with the patent preservation number CCTCC NO: M20221265 and the preservation time of August 9, 2022.

[0026] TM liquid medium: 10 g of proteose peptone, 1 g of casein hydrolysate, 5 g of glucose, 20 g of agar, 1000 ml of distilled water, and the pH is 5.0.

[0027] Soybean yellow broth: provided by the Institute of Agricultural Products Processing of Anhui Academy of Agricultural Sciences.

[0028] Malt: prepared according to the article “Determination of Yeast Growth Curve and Changes in Malt Sugar Content at Different Growth Times [J]. Veterinary Drugs and Feed Additives, 2006, 11 (1): 8-9.”

[0029] Soybean: commercially available, with the variety of Zhonghuang 13.

[0030] Method

[0031] The following methods are conventional methods known to those skilled in the art unless otherwise specified.

[0032] Preparation of fermentation yellow broth rooting bacteriostatic agent

[0033] 1.1. Preparation of seed liquid of each strain

[0034] According to the culture characteristics of the strain No. 1 and the strain No. 2, the activated strains were recovered, and then the activated and recovered strains were inoculated into TM liquid medium, and cultured at a temperature of 30 ℃ and a rotation speed of 100 r / min for 48 h. The above cultured bacterial liquid was inoculated into TM liquid medium at an inoculation amount of 2%, and cultured at a temperature of 30 ℃ and a rotation speed of 100 r / min until the viable bacterial count reached 10 8 / mL, to obtain the seed liquid of the strain No. 1 and the strain No. 2.

[0035] 1.2. Preparation of rooting bacteriostatic agent by fermentation yellow broth

[0036] The soybean yellow broth and the malt were stirred uniformly at a volume ratio of 4:1, and then filtered to obtain the fermentation substrate.

[0037] The seed liquid of the first strain and the second strain and the mixed seed liquid obtained by mixing the seed liquid of the first strain and the second strain in a volume ratio of 1:1 are inoculated into the fermentation medium respectively, the inoculation amount is 10% of the total volume of the fermentation medium, and after being mixed uniformly at a fermentation temperature of 30 DEG C for 72 h, the supernatant is obtained by filtration, and the supernatant is diluted by 300 times of water to obtain the bean sprout rooting inhibitor, which is recorded as rooting inhibitor A, rooting inhibitor B and rooting inhibitor C.

[0038] 2、Yellow bean sprout culture

[0039] The poor seeds (diseased seeds, defective seeds) and impurities are removed by artificial screening. 100 high-quality beans are selected in each treatment group, the beans are blanched and sterilized with water at 50 DEG C, and then the yellow beans are soaked in 25 DEG C water not more than twice the volume of the beans for 12 h. After 12 h, the water is drained, and the beans are evenly poured into the bean sprout machine for cultivation for 7 d. During the cultivation of the bean sprouts, the room temperature is maintained at about 25 DEG C, the relative humidity is 85%, and the cultivation is carried out in the dark.

[0040] 3、Rooting inhibitor treatment

[0041] According to the rooting inhibitor A-C, three treatment groups are set, and five repetitions are made. The rooting inhibitor is sprayed on each treatment group every 6 h.

[0042] In addition, a blank control group and a commercially available rooting agent group are set. The blank control group is sprayed with water every 6 h, and the commercially available rooting agent (plant growth hormone) is prepared by dissolving gibberellin in ethanol and then diluting it with 300 times of water. The concentration of the rooting agent is ensured to be equivalent to that of the rooting inhibitor (the use of 6-benzyladenine, 4-chlorophenoxyacetic acid sodium, gibberellin and other substances in the production process of bean sprouts is strictly prohibited according to the current regulations. This application is only used for experimental research). The single spraying amount of the above treatment groups is 10 ml.

[0043] Effect of different rooting inhibitor treatments on growth index of yellow bean sprouts

[0044] 4.1、Rooting and germination rate determination

[0045] Each group initially contains 100 yellow beans, and the number of seeds germinating / rooting is counted every 24 h until the end of the cultivation period (wherein, rooting and germination refers to the radicle emerging from the seed coat and exceeding the length of the bean by more than 1 cm). The average value is taken as the result;

[0046] Rooting and germination rate = number of rooting and germination beans / 100 x 100%.

[0047] 4.2、Bean sprout stem length, fresh weight and moisture content determination

[0048] (1) Stem length determination: randomly select 40 bean sprouts from each group, fully stretch the bean sprouts, and measure the stem length using a vernier caliper. Record the data and take the average (cm);

[0049] (2) Fresh weight determination: after 7 days of bean sprout cultivation, wash and drain the grown bean sprouts. Weigh the fresh weight of each group of bean sprouts on an analytical balance, mass (g / 100 seeds) = total mass / 100 x 100;

[0050] (2) Determination of water content: randomly select half of the bean sprouts from each group and record the fresh weight. Place the bean sprouts in an oven and dry at 55 ℃ for 1 day. Measure the dry weight of each group of bean sprouts after drying, water content = (fresh weight - dry weight) / fresh weight x 100%.

[0051] The results are shown in Table 1.

[0052] Table 1 Effect of different rooting bacteriostatic agents on the growth indicators of soybean sprouts

[0053] ;

[0054] From Table 1, it can be seen that during the cultivation of soybean sprouts, spraying rooting bacteriostatic agent compared with spraying water blank control group has a positive effect on improving the rooting germination rate, stem length, fresh weight and water content of bean sprouts. The rooting bacteriostatic agent obtained by fermentation of mixed bacteria is better than the single Lactobacillus paracasei or Kluyveromyces marxianus in improving the rooting germination rate, stem length, fresh weight and water content of bean sprouts, and is better than the common plant growth regulator - gibberellin.

[0055] 4.3 Effect of different rooting bacteriostatic agents on the disease resistance and rot resistance of soybean sprouts

[0056] The acid content in sprout tissue is low, and it is easy to be infected by microorganisms during cultivation and subsequent storage. The common bacteria on the surface of sprouts are Erwinia, Pseudomonas, Xanthomonas, enterobacter, etc. Because these bacteria have competitive advantage over pathogenic bacteria, they are the main rot-causing bacteria. It is of great significance to study the inhibitory effect of rooting bacteriostatic agent on bacteria during the cultivation of soybean sprouts to improve the disease resistance and rot resistance of soybean sprouts.

[0057] (1) Determination of total bacteria

[0058] When the culture is carried out to 3d, 20 samples of sprouted and rooted samples are selected from each group under sterile operation conditions according to GB / T 5009-96 “Food hygiene test method”, weighed and then diluted in 100 mL sterilized normal saline (concentration of 0.9 %), then 1 mL sterilized pipette is added into a test tube containing 9 mL normal saline for 10-fold incremental dilution, a suitable dilution is selected, and 1 mL is blown into a culture dish. The medium cooled to about 45 ℃ is poured into the dish and shaken evenly in a horizontal position, and after solidification, it is inverted in a 37±1 ℃ incubator for 24±1 h for colony counting. The number of colonies in the plate is calculated, multiplied by the dilution factor, and the total number of colonies per gram of sample (unit: CFU / g) is obtained.

[0059] (2) Disease rot rate statistics

[0060] After the culture of each group is completed, whether the soybeans or bean sprouts have phenomena such as rot, root rot, mold and plaque is observed and recorded, and the disease rot rate of the soybeans or bean sprouts = (number of soybeans or bean sprouts with disease rot phenomena) / 100 x 100 %, and the results of each group are averaged.

[0061] The results are shown in Table 2.

[0062] Table 2 Effect of different root inhibiting agents on the disease rot resistance of soybean sprouts

[0063] ;

[0064] As can be seen from Table 2, the root inhibiting agent obtained by fermentation of Kluyveromyces marxianus has a good antibacterial effect during the culture of bean sprouts, and is better than Lactobacillus paracasei. The total number of bacteria during the culture of soybean sprouts is less, resulting in the lowest disease rot rate of soybean sprouts, which is only 2.60 %. The root inhibiting agent obtained by fermentation of mixed bacteria also has a good antibacterial effect, and the total number of bacteria during the culture of soybean sprouts is 6.62 CFU / g, and the disease rot rate is 3.00 %. The secondary metabolites produced by the growth of Kluyveromyces marxianus can inhibit the growth and reproduction of spoilage bacteria during the growth of bean sprouts, and improve the disease resistance during the culture of bean sprouts.

[0065] 4.4 Effect of different root inhibiting agents on the content of harmful substances in soybean sprouts

[0066] 20 randomly selected bean sprouts from each group were used for determination of sulfite, nitrite and lead content, and the average value of each group was taken.

[0067] (1) Determination of sulfite: refer to the national standard “Determination of sulfur dioxide in food” (GB 5009.34-2022).

[0068] (2) Determination of nitrite: Refer to the second method of the national standard "National Food Safety Standard Determination of Nitrite and Nitrate in Food" (GB 5009.33-2016).

[0069] (3) Determination of lead content: Refer to the second method of the national standard "National Food Safety Standard Determination of Lead in Food" (GB5009.12-2017).

[0070] The results are shown in Table 3.

[0071] Table 3. Effects of different rooting and antibacterial agents on the content of harmful substances in soybean sprouts.

[0072] ;

[0073] As shown in Table 3, the rooting and antibacterial agents obtained by fermenting soybean whey and malt extract using mixed bacteria resulted in lower levels of sulfite, nitrite, and lead in the bean sprouts compared to those obtained by using only Kluyveromyces martensii or Lactobacillus paracasei. These levels were also lower than those in the blank control group. Furthermore, according to national standards, once the sulfite content exceeds 20 mg / kg... -1 A nitrite content exceeding 4 mg / kg is considered excessive. -1 This is considered exceeding the standard; the lead content exceeds 0.2 mg / kg. -1 The results of each group in this application were not considered to exceed the national regulations.

[0074] 4.5 Effects of different rooting and antibacterial agents on the nutritional indicators of soybean sprouts

[0075] Twenty bean sprouts were randomly selected from each group for the determination of protein, ascorbic acid, and SOD enzyme activity. The results were taken as the average value of each group.

[0076] (1) Protein determination: Refer to the first method of the national standard "National Food Safety Standard Determination of Protein in Food" (GB5009.5-2016).

[0077] (2) Determination of ascorbic acid: Refer to the third method of the national standard "National Food Safety Standard Determination of Ascorbic Acid in Food" (GB 5009.86—2016).

[0078] (3) Determination of SOD enzyme activity: The superoxide dismutase kit (catalog number: QS1500) from Shanghai Suoqiao Biotechnology Co., Ltd. was used for detection.

[0079] The results are shown in Table 4.

[0080] Table 4. Effects of different rooting and antibacterial agents on the nutritional indicators of soybean sprouts.

[0081] ;

[0082] As can be seen from Table 4, compared with the blank control group, the protein content of the bean sprouts cultivated by using the mixed bacteria as the strain for fermentation has no significant improvement, but the ascorbic acid concentration and the SOD enzyme activity in the bean sprouts are significantly improved, which makes the nutritional value and the antioxidant capacity of the bean sprouts improved, helps the bean sprouts resist environmental stress, the accumulation of nutritional ingredients is more abundant, and has a positive effect on the improvement of the quality of the bean sprouts.

[0083] The above-described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but should not be understood as a limitation on the scope of the patent of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application.

Claims

1. A yellow sap fermentation preparation, characterized in that, The yellow liquid fermentation preparation is made from Max Kluyveromyces (Yeast Inonotus maskarn). Kluyveromyces marxianus NNB-2 and Lactobacillus paracasei ( Lactobacillus paracasei LMX1 is obtained by inoculating a mixed culture into a fermentation substrate containing soybean slurry and malt extract and then fermenting it in a liquid state, wherein the volume ratio of soybean slurry to malt extract is 4:

1. The Lactobacillus paracasei ( Lactobacillus paracasei LMX1 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20211073, and the deposit date is August 24, 2021. The Max Kluyveromyces ( Kluyveromyces marxianus NNB-2 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M20221265, and the deposit date is August 9, 2022.

2. The yellow sap fermentation preparation according to claim 1, characterized in that, The Max Kluyveromyces ( Kluyveromyces marxianus NNB-2 and Lactobacillus paracasei ( Lactobacillus paracasei The mixing volume ratio of LMX1 is 1:

1.

3. The yellow sap fermentation preparation according to claim 1, characterized in that, The preparation method of the yellow sap fermentation agent includes the following steps: (1) Add Max Kluyveromycin ( Kluyveromyces marxianus NNB-2 and Lactobacillus paracasei ( Lactobacillus paracasei LMX1 seed culture was obtained by activation and resuscitation followed by liquid culture. (2) Add Max Kluyveromycin ( Kluyveromyces marxianus NNB-2 and Lactobacillus paracasei LMX1 ( Lactobacillus paracasei The seed liquid is mixed with the seed liquid and inoculated into the fermentation substrate at 10% of the total volume of the fermentation substrate. After mixing evenly, the mixture is fermented in liquid state at 30 ℃ for 72 h. The supernatant is then obtained by filtration.

4. A bean sprout rooting antibacterial agent, characterized in that, The bean sprout rooting antibacterial agent is obtained by diluting the yellow pulp fermentation preparation described in any one of claims 1-3 with 300-500 times the amount of water.

5. The application of the bean sprout rooting antibacterial agent as described in claim 4 in improving bean sprout growth indicators, characterized in that, The growth indicators include at least one of the following: rooting and germination rate, stem length, fresh weight, or water content.

6. The application of the bean sprout rooting antibacterial agent as described in claim 4 in promoting the disease resistance and rot resistance of bean sprouts.

7. The application of the bean sprout rooting antibacterial agent as described in claim 4 in reducing the content of harmful substances in bean sprouts, characterized in that, The hazardous substances include at least one of sulfites, nitrites, or lead.

8. The application of the bean sprout rooting antibacterial agent as described in claim 4 in promoting the nutritional indicators of bean sprouts, characterized in that, The nutritional indicators include at least one of protein content, ascorbic acid concentration, or SOD enzyme activity.