Bacterium-enzyme synergistic fermentation method for sweet potato residues

Abstract

The invention discloses a bacterium-enzyme synergistic fermentation method for sweet potato residues, which can realize rapid fermentation and has high protein content of the fermented sweet potato residues. According to the method, high-temperature-resistant alpha-amylase, cellulase and pectinase are used for carrying out enzymolysis on the sweet potato residues to decompose the sweet potato residues into reducing sugar, cellulose is preliminarily decomposed, and rapid and accurate starch conversion and cellulose degradation are carried out; a multi-bacterium combination of efficient degradation fibers, candida utilis capable of producing mycoprotein, lactobacillus plantarum JM113, bacillus subtilis and aspergillus niger is used for fermentation, so that the mycoprotein level in the fermented sweet potato residues is further improved, the nutritional value and the in-vitro digestibility are improved, the feeding price of the fermented sweet potato residues is improved, and the cost problem caused by high-moisture treatment is reduced; therefore, the economic value of the sweet potato residues is comprehensively improved, and the method has practical significance in waste utilization of the sweet potato residues, reduction of protein feed cost, alleviation of waste residue environmental pollution and improvement of industrial economic benefits. The method is suitable for popularization and application in the technical field of biological fermentation.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation, and specifically relates to a method for co-fermentation of fungus and enzyme for sweet potato residues. Background Art

[0002] Sweet potato, also known as Ipomoea batatas, is widely planted in China and is commonly used for food processing and starch production. As a by-product with high water content, difficult to transport and store after sweet potato starch production, sweet potato residues have caused serious damage to resource utilization and environmental friendliness. If scientifically developed and applied for feed, it can reduce the use level of cereal bran in the feed, and thus alleviate the problem of "competition for food between humans and livestock" in China. Existing fermentation technologies all have some disadvantages or problems. For example, in the patent with publication number CN114468127A, only through multi-fungus combined fermentation, the high moisture content of fresh sweet potato residues needs to be dried to make dry residues, and then water is added during fermentation, which will obviously greatly increase the production cost; and due to the high fiber content, the multi-fungus fermentation acts slowly and takes a long time, further leading to an increase in cost, which is not conducive to large-scale treatment and industrial production. The patent with publication number CN116898040A also only uses multi-fungus fermentation, with a long time and poor effect. At the same time, the protein content of sweet potato residues after fermentation by the above two methods is not high, and the nutritional value and feed value of sweet potato residues are not high. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for co-fermentation of fungus and enzyme for sweet potato residues that can ferment quickly and has a relatively high protein content in sweet potato residues after fermentation.

[0004] The technical solution adopted by the present invention to solve its technical problems is as follows: The method for co-fermentation of fungus and enzyme for sweet potato residues includes the following steps:

[0005] A. Enzymatic hydrolysis: Use a multi-enzymatic hydrolysis combination of thermotolerant α-amylase Amy, cellulase Cel, and pectinase Xyl to perform enzymatic hydrolysis on sweet potato residues; the enzymatic hydrolysis combination and parameters are: Amy 130 - 150U / g, Cel 230 - 260U / g, Xyl 20 - 40U / g, temperature 40 - 55; pH 3 - 6; time 5 - 8h;

[0006] B. Preparation of the culture medium: Mix the sweet potato residues, bran, urea, and ammonium sulfate treated in step A in a certain proportion to obtain the culture medium; the mass ratio of the sweet potato residues to the bran is (2.5 - 3.5):1, the addition amount of urea is 3 - 5% of the total mass of the sweet potato residues and the bran, and the addition amount of ammonium sulfate is 3 - 5% of the total mass of the sweet potato residues and the bran;

[0007] C. Fermentation: Add a multi-strain combination of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger to the culture medium for fermentation treatment; the addition amounts of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger are all 1-3% of the mass of the culture medium, and the concentrations of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger are all 1×10 8 cfu / mL, the fermentation time is 3-5 days, and the fermentation temperature is 25-35°C. Lactobacillus plantarum JM113 was deposited at the China Center for Type Culture Collection (CCTCC) on November 18, 2015. Deposit address: Wuhan University, Wuhan, China, Zip Code 430072; Deposit number: CCTCC NO: M2015680.

[0008] Furthermore, in step A, the enzyme combination and enzyme hydrolysis parameters are Amy 141.3U / g, Cel 247.5U / g, Xyl 27.1U / g, temperature 46.5°C; pH 5.1; time 6.4h.

[0009] Furthermore, in step B, the mass ratio of sweet potato residue to bran is 3:1, the addition amount of urea is 4% of the total mass of sweet potato residue and bran, and the addition amount of ammonium sulfate is 4% of the total mass of sweet potato residue and bran.

[0010] Furthermore, in step C, the addition amounts of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger are all 2% of the mass of the culture medium, the fermentation time is 4 days, and the fermentation temperature is 31°C.

[0011] Furthermore, the deposit number of Lactobacillus plantarum JM113 is CCTCC NO: M2015680.

[0012] Furthermore, the cultivation method of Lactobacillus plantarum JM113 includes the following steps: Using the chicken digestive tract contents and mucosa as the sample source, perform 10-fold serial dilutions up to 10 -7 , take 200 μl from each dilution gradient and spread it on MRS solid medium, place it in an incubator at 37°C and incubate it upside down for 48 h, and preserve it after purification by picking colonies and streaking.

[0013] The beneficial effects of the present invention are as follows: The method for the combined fermentation of bacteria and enzymes for sweet potato residues makes full use of the characteristics that fresh sweet potato residues have high moisture content and are suitable for enzymatic hydrolysis. By using thermotolerant α-amylase, cellulase, and pectinase, the sweet potato residues are first enzymatically hydrolyzed into reducing sugars, and cellulose is preliminarily decomposed, enabling rapid and precise starch conversion and cellulose degradation. This can reduce viscosity and increase the contact area between the bacteria and enzymes, and the reducing sugars further serve as the nutrient source for the bacteria, improving the nutrient source and reproduction area of the bacterial population, and providing a more favorable survival and reproduction environment for the subsequent fermenting bacteria. Subsequently, through the use of a multi-bacteria combination of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger, which can efficiently degrade fibers and produce bacterial proteins, the anti-nutritional factors are further decomposed through bacterial fermentation to produce bacterial proteins, further increasing the level of bacterial proteins in the fermented sweet potato residues, improving the nutritional value and in vitro digestibility, enhancing its feeding value, and reducing the cost problem caused by high-moisture treatment. Furthermore, the economic value of sweet potato residues is comprehensively improved, which has practical significance for the waste utilization of sweet potato residues, reducing the cost of protein feed, alleviating the environmental pollution of waste residues, and improving the economic benefits of the industry.

[0014] The Lactobacillus plantarum JM113 was deposited at the China Center for Type Culture Collection (CCTCC) on November 18, 2015. The deposit address is: Wuhan University, Wuhan, China, Zip Code 430072; the deposit number is CCTCC NO: M2015680. Description of the Drawings

[0015] Figure 1 It is a graph for the in vitro evaluation of the antioxidant activities of different lactic acid bacteria;

[0016] Figure 2 It is an infrared spectrum graph of enzymatically hydrolyzed sweet potato residues;

[0017] Figure 3 It is a scanning electron microscope graph of sweet potato residues before and after enzymatic hydrolysis;

[0018] Figure 4 It is a graph showing the effects of sweet potato residues before and after enzymatic hydrolysis on water absorption capacity, swelling capacity, and oil holding capacity;

[0019] Figure 5 It is a graph of the dry matter digestibility of sweet potato residues in vitro simulating 14-day-old and 35-day-old poultry;

[0020] Figure 6 It is a graph of the true protein digestibility of sweet potato residues in vitro simulating 14-day-old and 35-day-old poultry;

[0021] Figure 7 It is a graph of the dry matter and true protein digestibility of sweet potato residues in vitro simulating different-day-old poultry. Detailed Embodiments

[0022] Since the water content in sweet potato residue is as high as 90%, other main components also include starch, cellulose, hemicellulose, pectin, etc. For the purpose of rapidly degrading fibers and improving fermentation ability, this invention selects thermotolerant α-amylase for decomposing starch and reducing viscosity, cellulase for decomposing cellulose, and pectinase for degrading pectin which has an adhesion effect and hinders enzymatic hydrolysis and fermentation. At the same time, using sweet potato residue as raw material to produce microbial protein can not only increase the protein content in feed, but also supplement the amino acid level to improve the protein quality. Meanwhile, during the fermentation process, microorganisms can secrete various hydrolases such as cellulase, amylase and protease, which can rapidly degrade macromolecular substances (fibers, starch, etc.) in sweet potato residue, improving the nutritional level and feed digestibility. Therefore, on the basis of enzymatic hydrolysis, taking the content of true protein in the fermentation product as an index, referring to common feed fermentation strains with high microbial protein synthesis efficiency, which can improve the nutritional value and digestion efficiency of substrates and are safe and harmless, and screening out the single strain with the best fermentation of sweet potato residue to produce microbial protein, multiple groups of sweet potato residue are fermented, and through grey theory correlation analysis, the optimal strain combination for fermenting sweet potato residue is obtained as: Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, Aspergillus niger. Thus, the method for co-fermentation of bacteria and enzymes for sweet potato residue described in this invention is obtained. Specifically, it includes the following steps:

[0023] A. Enzymatic hydrolysis: Using a multi-enzymatic hydrolysis combination of thermotolerant α-amylase Amy, cellulase Cel and pectinase Xyl to conduct enzymatic hydrolysis treatment on sweet potato residue; the enzymatic hydrolysis combination and parameters are: Amy 130 - 150U / g, Cel 230 - 260U / g, Xyl 20 - 40U / g, temperature 40 - 55; pH 3 - 6; time 5 - 8h;

[0024] B. Preparation of culture medium: Mixing the sweet potato residue, wheat bran, urea and ammonium sulfate treated in step A in a certain proportion to obtain the culture medium; the mass ratio of sweet potato residue to wheat bran is (2.5 - 3.5):1, the addition amount of urea is 3 - 5% of the total mass of sweet potato residue and wheat bran, and the addition amount of ammonium sulfate is 3 - 5% of the total mass of sweet potato residue and wheat bran;

[0025] C. Fermentation: Adding a multi-strain combination of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis and Aspergillus niger to the culture medium for fermentation treatment; the addition amounts of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis and Aspergillus niger are all 1 - 3% of the mass of the culture medium, and the concentrations of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis and Aspergillus niger are all 1×10 8 cfu / mL, the fermentation time is 3 - 5d, and the fermentation temperature is 25 - 35℃.

[0026] In the above embodiments, the optimal enzyme combination and optimal enzymatic hydrolysis parameters obtained after optimization by the response surface method are Amy 141.3 U / g, Cel 247.5 U / g, Xyl 27.1 U / g, temperature 46.5 °C; pH 5.1; time 6.4 h. Further, in step B, the mass ratio of the sweet potato residue to the bran is preferably 3:1, the addition amount of urea is preferably 4% of the total mass of the sweet potato residue and the bran, and the addition amount of ammonium sulfate is preferably 4% of the total mass of the sweet potato residue and the bran.

[0027] To achieve the best fermentation effect, in step C, the addition amounts of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger are all 2% of the mass of the culture medium, the fermentation time is 4 d, and the fermentation temperature is 31 °C. Specifically, the optimal true protein fermentation conditions are an inoculation amount of 1.9%, a fermentation time of 4.5 d, and a fermentation temperature of 31.9 °C; the sweet potato residue fermentation conditions with the highest acid protease activity are an inoculation amount of 2.1%, a fermentation time of 4 d, and a fermentation temperature of 30.3 °C; the sweet potato residue fermentation conditions with the highest neutral protease activity are an inoculation amount of 2.0%, a fermentation time of 4 d, and a fermentation temperature of 32.2 °C.

[0028] Among them, JM113 is a preserved strain screened and continuously studied by the laboratory itself, and the other three are market mainstream product strains. Lactobacillus plantarum JM113 was preserved in the China Center for Type Culture Collection (CCTCC) on November 18, 2015. Preservation address: Wuhan University, Wuhan, China, Zip Code 430072; Preservation number: CCTCC NO: M2015680.

[0029] Lactobacillus plantarum JM113 has the advantages of high fermentability, strong antioxidant performance, and strong pathogenic bacteria inhibitory effect. By using JM113 in the fermentation process, the fermentation effect of the substrate can be improved, the number of harmful bacteria can be reduced, and the nutritional value and feed safety of the fermented sweet potato residue can be improved.

[0030] The cultivation method of Lactobacillus plantarum JM113 includes the following steps: Using the chicken digestive tract contents and mucosa as the sample source, performing 10-fold serial dilutions to dilute to 10 -7 , taking 200 μl of each dilution gradient and spreading it on MRS solid medium, placing it in an incubator at 37 °C for inverted culture for 48 h, and preserving it after purification by picking colonies and streaking.

[0031] The acid and bile salt tolerance properties of Lactobacillus plantarum JM113 are shown in Table 1. After culturing in MRS medium with pH 2.5 and bile salt concentration of 0.25% for 2 h, the viable cell count hardly decreased, and the order of magnitude remained at 108, indicating that the strain has extremely strong stress resistance.

[0032] Table 1 Acid and Bile Salt Tolerance Properties of Lactobacillus plantarum JM113

[0033]

[0034] Lactobacillus plantarum JM113 can grow abundantly under the conditions of pH 6.5, temperature 37°C, and rotation speed 200 rpm / min. The fermentation broth is centrifuged at low temperature and high speed to obtain the bacterial cells, which are freeze-dried for 24 hours and ground into bacterial powder. The viable count of each gram of bacterial powder is as high as 10 10 cfu. As shown in Table 2, the diameters of the inhibition zones of Lactobacillus plantarum against intestinal pathogenic Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, Pasteurella, and Staphylococcus aureus can reach 17 - 20 mm.

[0035] Table 2 Inhibition Zone Diameters of Lactobacillus plantarum JM113 (mm)

[0036] Treatment Salmonella typhimurium Pasteurella Escherichia coli K88 Staphylococcus aureus Salmonella enteritidis Lactobacillus plantarum 19.65±0.36 18.24±0.32 17.24±0.26 17.47±0.14 17.10±0.08 MRS 0 0 0 0 0

[0037] Lactobacillus plantarum JM113 also has strong antioxidant properties. Figure 1 For the in vitro evaluation of the antioxidant activities of different lactic acid bacteria, among them, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (A), hydroxyl radical scavenging activity (B), superoxide anion radical scavenging activity (C), and reducing activity (D). CFE: cell-free extract; DBS: dead strain; LBS: live strain. As Figure 1 shown, compared with other strains, its DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide anion radical scavenging activity, and reducing activity are all significantly higher than the other two strains.

[0038] Example

[0039] Sweet potato residue was purchased from farmers on Yimutian. First, Amy 141.3 U / g, Cel 247.5 U / g, and Xyl 27.1 U / g were added to the sweet potato residue, and the enzymatic hydrolysis parameters were temperature 46.5°C; pH 5.1; time 6.4 h; then the enzymatically hydrolyzed sweet potato residue was mixed with bran at a mass ratio of 3:1, and 4% of the total mass of sweet potato residue and bran of urea and 4% of the total mass of sweet potato residue and bran of ammonium sulfate were added to obtain the medium; finally, Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger accounting for 2% of the medium mass were added respectively, and fermentation was carried out for 4 d at a fermentation temperature of 31°C.

[0040] Each index and the detection method are as follows:

[0041] 1.1 Determination of sweet potato residue components, as shown in Table 3:

[0042] Table 3

[0043]

[0044] 1.2 Determination of the amount of reducing sugar produced:

[0045] Accurately weigh 0.5 g of the sample according to the above steps and put it into a 50 mL beaker. First, use a small amount of distilled water to make the sample into a paste, then add 20 mL of distilled water and stir evenly. Place the beaker in a 50 °C constant temperature water bath for 30 min to leach out the reducing sugar. Then transfer the leaching solution (including the precipitate) to a 50 mL centrifuge tube and centrifuge at 4000 rpm for 5 min. Wash the precipitate once with 20 mL of distilled water and then centrifuge again. Collect the supernatant of the second centrifugation in a 100 mL volumetric flask, make up to the mark with distilled water, mix well, and it can be used as the reducing sugar solution to be measured. Detect its OD value at a wavelength of 540 nm.

[0046] Amount of reducing sugar produced (mg / g) = (C × VT) / (m × VS) × 100

[0047] Where: C = amount of sugar (mg) found from the standard curve

[0048] VT = volume of the extraction solution (mL)

[0049] m = mass of the plant sample (mg)

[0050] VS = volume of the sample used during the determination (mL)

[0051] Determination of the contents of plant starch, pectin and soluble sugar:

[0052] Use a biochemical detection kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) for detection. All the determination steps and calculation formulas are carried out strictly in accordance with the kit instructions.

[0053] 1.3 Determination of true protein

[0054] Crush the feed sample with a feed sample crusher, pass it through a 40-mesh sieve, accurately measure about 0.5 g of the sample and put it into a 250 mL container. Then add 75 mL of distilled water to it, and then heat the mixture to the boiling state. After boiling for 30 min, add 20 mL of 2.5% sodium hydroxide solution and 20 mL of 10% copper sulfate solution to the solution, and stir thoroughly with a glass rod until uniform. Subsequently, boil gently for a period of time, and after cooling, let it stand for more than 2 h. Screen through qualitative filter paper, and then rinse the precipitate with hot water at 70 to 80 °C for more than five times. Use five drops of 5% barium chloride solution and a small amount of hydrochloric acid to detect the filtrate until no more white barium sulfate precipitate is produced. Dry the precipitate and the filter paper in an oven at 65 °C.

[0055] 1.4 Determination of soluble protein

[0056] Accurately weigh 1.0000 g of bovine serum albumin and prepare a 1 mg / mL solution; weigh 0.1 g of Coomassie Brilliant Blue G-250, add 50 mL of 95% ethanol, then add 100 mL of 85% (weight / volume) phosphoric acid, and finally make up the volume to 1000 mL with distilled water. Accurately weigh about 0.1 g of feed sample (passed through a 40-mesh sieve), add 2 mL of pre-cooled phosphate buffer with a pH of 7.0, and add zirconia beads for grinding and homogenization. Centrifuge at 4 °C and 10,000 r / min for 10 min, and aspirate the supernatant for standby. Dilute the 1 mg / mL standard solution stepwise to 0.5, 0.25, and 0.125 mg / mL. Accurately aspirate 1 mL of the solution and place it into a 10-mL test tube respectively, mix it with 5 mL of Coomassie Brilliant Blue reagent, let it stand for 2 min, then measure the absorbance at 595 nm, and draw a standard curve. Aspirate 1 mL of the sample extract, add 5 mL of Coomassie Brilliant Blue reagent, mix well, let it stand for 2 min, then measure the absorbance of the sample at 595 nm, and calculate the content of soluble protein in the sample according to the standard curve.

[0057] 1.5 Fourier transform infrared spectroscopy determination:

[0058] Take an appropriate amount of the sample. After conventional drying, grind and press it into tablets together with potassium bromide (KBr should be ground to less than 200 mesh before use, dried at above 120 °C for more than 4 h and then placed in a desiccator for standby. If caking is found, it should be redried. The prepared empty KBr should be transparent, and the transmittance should be above 75% compared with air. KBr is of chromatographic grade). Press it into a thin slice with a diameter of 13 mm and a thickness of 0.5 mm. The ratio of potassium bromide to the sample is 100:1 (about 1 - 2 mg of the sample). Analyze it with a Fourier transform infrared spectrometer (spectral range: 400 - 4,000 cm-1; resolution: 0.16 cm-1; absorption accuracy: 0.1% T; wavenumber accuracy: better than 0.01 cm-1; signal-to-noise ratio: 55,000:1). The resolution is 4 cm-1, the number of scans is 32 times, and the scanning range is 650 - 4000 cm-1.

[0059] 1.6 Scanning electron microscopy analysis:

[0060] Freeze-dry the sample, spray gold on the surface, and then observe and analyze it using a desktop scanning electron microscope (Hitachi TM3000, Japan).

[0061] 1.7 Physical and chemical property analysis:

[0062] (1) Determination of water absorption

[0063] The method of Khuthadzo Ngoma et al. (2019) was adopted to measure the water absorption capacity of sweet potato residue before and after enzymatic hydrolysis. Weigh approximately 1 g of the sample in a conical graduated centrifuge tube and disperse it in 10 mL of water. The mixture was shaken vigorously for 1 min at room temperature. After the sample was allowed to stand for 30 min, it was centrifuged at 5,000×g for 30 min. The volume of free water was directly measured from the centrifuge tube.

[0064] Water absorption capacity (mL / g) = (V0 - V) / m

[0065] Where: m = mass of the weighed sample (g)

[0066] V = volume of free water after centrifugation (mL)

[0067] V0 = volume of added water (10 mL)

[0068] (2) Determination of swelling force

[0069] The method of Anyasi et al. (2017) was used to determine the swelling capacity of sweet potato residue samples before and after enzymatic hydrolysis, that is, approximately 1 g of the sample was mixed with 10 mL of distilled water in a centrifuge tube, heated at 80 °C for 30 min with continuous stirring. The centrifuge tube was taken out and cooled to room temperature. After cooling, the sample was centrifuged at 2200 rpm for 15 min. The supernatant was decanted, and then the weight of the slurry was measured.

[0070] Swelling force (g / g) = M / m

[0071] Where: m = mass of the weighed sample (g)

[0072] M = weight of the slurry after centrifugation (g)

[0073] (3) Determination of oil-holding capacity

[0074] Accurately weigh 3 g (M1) of the sample (on a dry basis) into a centrifuge tube (M0), add 30 mL of soybean salad oil, stir evenly, let it stand at 37 °C for 1 h, centrifuge at 4000 rpm for 15 min, and discard the upper layer of oil. Wipe dry the grease and moisture adhering to the inner and outer walls of the centrifuge tube, and weigh the mass of the centrifuge tube as M2.

[0075] Oil-holding capacity (g / g) = (M2 - M1 - M0) / M1

[0076] Where: M0 = mass of the centrifuge tube (g)

[0077] M1 = mass of the weighed sample (g)

[0078] M2 = mass of the centrifuge tube after removing the upper layer of oil (g)

[0079] 1.8 In vitro simulated digestion test:

[0080] The enzymatic hydrolysis of sweet potato residue was subjected to a conventional in vitro digestion simulation. The in vitro digestion test was determined using (L. Stokvis et.al 2021). 1 g of the sample was incubated with 75 mL of 0.1 M phosphate buffer solution (pH 6.0) and 0.2 M HCl solution until the pH reached 2.0. 1 mL of pepsin solution (25 g / L, 2000 U / g) was added, and the sample was incubated at 39 °C with continuous stirring for 2 h. Thereafter, 30 mL of 0.2 M phosphate buffer (pH 6.8) and NaOH were added until the pH reached 6.8. 1 mL of pancreatin 100 g / L was added, and incubation was continued under the same conditions for 4 h. The in vitro incubation was terminated after 2 h, 4 h, or 6 h, representing gastric and small intestine digestion, respectively. After the in vitro digestion was completed, the biomimetic digestion residue was collected.

[0081] For fermented sweet potato residue, the in vitro simulated digestion and absorption of the fermentation products of sweet potato residue were further evaluated with reference to the literature (Chuang et al. 2020; Zhang Tieying 2002). A two-step determination method was used with some modifications to the method, and the pH was adjusted with HCl or NaOH solution. To simulate gastric juice, 5.18 g of sodium chloride, 0.50 g of potassium chloride, and 15.60 g of sodium dihydrogen phosphate were weighed and dissolved in distilled water to 1000 mL. (0.465 g of pepsin was added to simulate the gastric juice of 14-day-old infants, and 0.7020 g of pepsin was added to simulate the gastric juice of 35-day-old infants), pH 2.66 at 41 °C, and refrigerated at 4 °C. To simulate intestinal juice, 13.68 g of anhydrous disodium hydrogen phosphate and 50.44 g of sodium dihydrogen phosphate were dissolved in distilled water and adjusted to 1000 mL (0.7770 g of trypsin was added to simulate the intestinal juice of 14-day-old infants, and 0.9505 g of trypsin was added to simulate the intestinal juice of 35-day-old infants), pH 6.03, 41 °C; stored at room temperature.

[0082] Simulated gastric digestion stage: Weigh 1 g of the sample into a 50 mL sterile and enzyme-free centrifuge tube. Then, add 8 mL of freshly prepared gastric juice. While shaking, quickly add 1 mL of appropriately diluted exogenous enzyme, and react the mixture in a 41 °C water bath for 2 h.

[0083] Simulated small intestine digestion stage: Take out the sample from the gastric simulation digestion stage, add 6 mL of intestinal juice, and incubate the test tube in the same shaking water bath at 41 °C for 6 h. Samples were taken every 2 h during this period. After digestion was completed, all samples were placed in an ice bath to terminate the enzyme action. After centrifugation (5000 g, 10 min), the supernatant was stored at 4 °C. The undigested residue was washed three times with distilled water and then dried, and its dry matter, true protein, and amino acid digestibility were detected.

[0084] Dry matter digestibility (%) = (Initial mass of sample - Residual mass after digestion) / Initial mass of sample

[0085] True protein digestibility (%) = (Initial true protein content of sample - True protein content of residual sample after digestion) / Initial true protein content of sample

[0086] Amino acid digestibility (%) = (Initial amino acid content of sample - Amino acid content of residual sample after digestion) / Initial amino acid content of sample

[0087] 1.9 Statistical methods

[0088] The experimental data were initially recorded and organized using Microsoft Excel 2017, and then the independent samples t-test (two-tailed) and one-way analysis of variance (one-way ANOVA) were performed on the data using the IBM SPSS Statistics 26.0 statistical software, and the Duncan's method was used for multiple comparisons. P ≤ 0.05 was considered significantly different, P ≤ 0.01 was considered extremely significantly different, and 0.05 < P ≤ 0.10 was considered to have a significant trend. All results in the statistical methods were presented as mean ± SEM, and the data result graphs were drawn using GraphPad Prism 9.2.

[0089] The test results are as follows:

[0090] 2.1 Enzymatic hydrolysis process:

[0091] Amy: 124.8 U / g, Cel: 249.3 U / g, Xyl: 22.6 U / g; temperature 46.5 °C, pH 5.1, 6.4 h, and the reducing sugar reached 257.9 mg / g. The wet residue increased by 14.3% compared with the optimized dry residue, and increased by 347.5% and 411.3% respectively for the dry and wet residues compared with the original pulp group;

[0092] The enzymatic hydrolysis process increased the reducing sugar level of sweet potato residues. The original sugar contents after enzymatic hydrolysis were 211.3 mg / g (dry residue) and 250.1 mg / g (wet residue) respectively. The wet residue increased by 18.3% compared with the optimized dry residue, and increased by 347.5% (dry residue) and 411.3% (wet residue) respectively compared with the original pulp group (60.8 mg / g).

[0093] The nutritional components of sweet potato residues before and after enzymatic hydrolysis are shown in Table 4. The results showed that the enzymatic hydrolysis process significantly changed the nutritional composition of sweet potato residues. After enzymatic hydrolysis, the crude protein and crude fat contents of sweet potato residues increased significantly, while the levels of NDF, ADF, starch and pectin decreased significantly (P < 0.05). Among them, the crude protein and crude fat levels of the wet residue after enzymatic hydrolysis (SM) were significantly higher than those of the dry residue after enzymatic hydrolysis (GM), and the degradation effects of NDF and ADF were also better (P < 0.05); there was no significant difference in the starch and pectin contents between the two groups.

[0094] Table 4 Nutritional value evaluation of sweet potato residue before and after enzymatic hydrolysis

[0095]

[0096] The results of infrared spectroscopy are as Figure 2 shown; the infrared absorption spectra of sweet potato residue before and after enzymatic hydrolysis are similar, with characteristic absorption peaks of cellulose. In different treatment groups, as the vibration band gradually increases, the absorbance from bottom to top is the original pulp group - dry residue CON - wet residue CON - dry residue enzymatic hydrolysis group (GM) - wet residue enzymatic hydrolysis group (SM). Among them, compared with the original pulp group, the wave peaks after enzymatic hydrolysis optimization of both wet and dry residues are significantly higher than those of the untreated group, and the wave peak of the SM group after enzymatic hydrolysis is higher than that of the GM group.

[0097] The scanning electron micrographs of sweet potato dry / wet residue CON group and the samples after enzymatic hydrolysis are as Figure 3 shown. Before enzymatic hydrolysis of sweet potato residue, starch and pectin are tightly bound, while after enzymatic hydrolysis, the fibrous substances on the surface of sweet potato residue are significantly reduced, becoming smoother and looser, indicating that the enzymatic hydrolysis process destroys the tight meshing state of starch and cellulose.

[0098] The analysis results of the physical and chemical properties of sweet potato residue before and after enzymatic hydrolysis are as Figure 4 shown. After enzymatic hydrolysis of sweet potato residue, the water absorption capacity, swelling capacity, and oil holding capacity are all improved compared with the original pulp. Among them, the wet residue state of enzymatically hydrolyzed and optimized sweet potato residue has the highest water holding rate, swelling capacity, and oil holding capacity, which are 2.55 mL / g, 6.23 g / g, and 1.35 g / g respectively, increasing by 47%, 26.3%, and 82% compared with the CON group, and increasing by 39%, 17%, and 36% compared with the original pulp group.

[0099] The enzymatic hydrolysis process of sweet potato residue significantly changes the nutritional value, structural characterization, physical and chemical properties, and in vitro digestibility of sweet potato residue. Specifically, the contents of NDF, ADF, and starch decrease significantly, the physical structure becomes loose, and the enzymatic hydrolysis process enhances the water absorption capacity, swelling capacity, and oil holding performance of sweet potato residue, which is helpful for the subsequent fermentation reaction.

[0100] 2.2 Fermentation process:

[0101] As can be seen from the results in Tables 5 and 6, after fermenting enzymatically hydrolyzed sweet potato residues (with an addition amount of 2%, a fermentation time of 4 days, and a fermentation temperature controlled at 31°C), the true protein content in the fermentation product is 9.13%, the acid protease activity is 1191.3 U / g, and the neutral protease activity is 1011.3 U / g. Compared with the original sweet potato residues, the true protein in the fermented product increased by 157.9%, the acid protease activity increased by 1740.8%, and the neutral protease activity increased by 1913.0%. In addition to the increase in true protein and protease activity, the NDF, ADF, and toxins in the fermented sweet potato residues also decreased compared with the original sweet potato residue group. According to the amino acid analysis results, the contents of various amino acids all increased.

[0102] Table 5 Comparison of nutritional components of sweet potato residues before and after fermentation

[0103]

[0104] Table 6 Comparison of amino acid contents in the products before and after fermentation

[0105]

[0106]

[0107] 2.3 In vitro digestion test:

[0108] 2.3.1 Enzymatic hydrolysis

[0109] The results of the in vitro digestion test of enzymatically hydrolyzed sweet potato residues are shown in Table 7. During the enzymatic hydrolysis process, the in vitro dry matter digestibility of sweet potato dry and wet residues increased significantly (P<0.01). At 2 h, compared with the blank group and the original pulp group, the digestibility of the GM group increased by 205.56% and 51.41% respectively; the SM group increased by 177.65% and 37.59% respectively compared with the blank group and the original pulp group. As the culture time increased, the dry matter digestibility of sweet potato dry and wet residues further increased.

[0110] Table 7 Effects of sweet potato residues before and after enzymatic hydrolysis on dry matter digestibility

[0111]

[0112] Note: Different lowercase letters on the right shoulder of the same column indicate significant differences (P<0.05); CON: Original sweet potato residues without any processing; PRO: Using the same process as the enzymatic hydrolysis group, but without adding the corresponding enzyme; GM: Optimized enzymatic hydrolysis in dry basis state; SM: Optimized enzymatic hydrolysis in wet basis state

[0113] Table 8 shows the effect of enzymatic hydrolysis of sweet potato residues on the production of reducing sugars; the results in Table 8 indicate that the enzymatic hydrolysis group after in vitro digestion significantly increased the production of reducing sugars in wet and dry sweet potato residues (P<0.01). At 2 h, compared with the blank group, the reducing sugar yield of the GM group increased by 654.47%, and compared with the original pulp group, it increased by 50.28%; the SM group increased by 603.26% compared with the blank group and by 40.08% compared with the original pulp group. As the culture time increased, the production of reducing sugars in wet and dry sweet potato residues gradually decreased.

[0114] Table 8 Effect of enzymatic hydrolysis of sweet potato residues on the production of reducing sugars

[0115]

[0116]

[0117] Note: Different lowercase letters in the right shoulder of the same column indicate significant differences (P<0.05); CON: raw potato residues without any processing; PRO: using the same process as the enzymatic hydrolysis group, but without adding the corresponding enzyme; GM: optimized enzymatic hydrolysis in dry basis; SM: optimized enzymatic hydrolysis in wet basis

[0118] 2.3.2 Fermentation

[0119] The enzymatically hydrolyzed sweet potato residues were fermented, and the digested products after fermentation were subjected to in vitro simulated digestion tests of broiler chicken intestines, as Figure 5 and Figure 5 shown. Figure 5 and Figure 6 The results in

[0120] showed that during the simulated digestion process of 14-day-old broiler chickens, the digestibility of fermented sweet potato residues was significantly higher than that of unfermented sweet potato residues during the gastric digestion stage and the small intestine digestion stage (P<0.05). During the simulated digestion process of 35-day-old broiler chickens, the digestibility during the first 4 h of the gastric digestion stage and the small intestine digestion stage was significantly higher than that of unfermented sweet potato residues. From the results of true protein digestibility, it was found that whether in the in vitro simulated environment of 14-day-old or 35-day-old broiler chickens, the true protein digestibility of the fermentation group was significantly higher than that of the non-fermentation group during the gastric digestion stage. At the 14-day-old stage, the true protein digestibility of the fermentation group during the first 2 h of small intestine digestion was significantly higher than that of the non-fermentation group (P<0.05). At the 35-day-old stage, the true protein digestibility of the fermentation group 4 h after small intestine digestion was significantly higher than that of the non-fermentation group (P<0.05).

[0120] Taking the digestion results of the two stages together, as Figure 7 shown in Figure 7 , during the gastric digestion stage and the first 2 h and 4 h of small intestine digestion, the dry matter digestibility of 35-day-old broilers was significantly higher than that of 14-day-old broilers (P<0.05), and there was no significant difference in dry matter digestibility after 4 h (P>0.05); while there was no significant difference in true protein digestibility between the two ages (P>0.05).

[0121] The results of amino acid digestibility are shown in Tables 9 and 10. The digestibility of 17 hydrolyzed amino acids in fermented sweet potato residue is generally higher than that in the unfermented group. In the simulated digestion of 14-day-old, the digestibility of 17 amino acids in fermented sweet potato residue is generally above 60%. In the simulated digestion of 35-day-old, the amino acid digestibility of fermented sweet potato residue is above 70%. Among them, the digestibility of threonine, serine, tyrosine, lysine and proline reaches above 80%, and the digestibility of aspartic acid and methionine reaches above 90%.

[0122] Table 9 Amino acid digestibility in the in vitro simulated digestive tract of 14-day-old poultry

[0123]

[0124]

[0125] Table 10 Amino acid digestibility in the in vitro simulated digestive tract of 35-day-old poultry

[0126]

[0127]

[0128] As can be seen from the above, the enzymatic hydrolysis process significantly changes the nutritional value, structural characterization, physical and chemical properties and in vitro digestibility of sweet potato residue. Specifically, the contents of NDF, ADF and starch decrease significantly, the physical structure becomes loose, and the enzymatic hydrolysis process enhances the water absorption, swelling and oil-holding properties of sweet potato residue, increasing its in vitro digestibility. The subsequent fermentation process further improves the dry matter digestibility of sweet potato residue. The true protein digestibility is significantly higher than that of the non-fermented group in some stages, and the amino acid digestibility is above 70%, further improving the nutritional value of sweet potato residue and providing feasibility for using fermented sweet potato residue as protein feed.

[0129] The method for co-fermentation of fungus and enzyme for sweet potato residues fully utilizes the characteristics that fresh sweet potato residues have high moisture content and are suitable for enzymatic hydrolysis. By using thermotolerant α-amylase, cellulase and pectinase to hydrolyze sweet potato residues into reducing sugars first, initially decomposing cellulose, rapid and precise starch conversion and cellulose degradation can be carried out, which can reduce viscosity and increase the contact area between the fungus and enzyme and the substrate. And the reducing sugars therein further serve as the nutrient source of the fungus, improving the nutrient source and reproduction area of the bacterial population, and providing a more favorable living and reproduction environment for the subsequent fermenting bacteria; Subsequently, through the use of a multi-bacteria combination of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis and Aspergillus niger that can efficiently degrade fibers and produce microbial protein for fermentation, the anti-nutritional factors are further decomposed by the strain fermentation to produce microbial protein, further increasing the level of microbial protein in the fermented sweet potato residues, improving the nutritional value and in vitro digestibility, enhancing its feeding value, and reducing the cost problem brought by high-moisture treatment. Furthermore, the economic value of sweet potato residues is comprehensively improved, which has practical significance for the waste utilization of sweet potato residues, reducing the cost of protein feed, alleviating the environmental pollution of waste residues, and improving the economic benefits of the industry.

[0130] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for co-fermentation of sweet potato residues by enzymes and microorganisms, characterized in that It includes the following steps: A. Enzymatic hydrolysis: The sweet potato residue is subjected to enzymatic hydrolysis treatment using a multi-enzymatic hydrolysis combination of thermotolerant α-amylase Amy, cellulase Cel, and pectinase Xyl. The enzymatic hydrolysis combination and parameters are as follows: Amy 130 - 150 U / g, Cel 230 - 260 U / g, Xyl 20 - 40 U / g, temperature 40 - 55°C; pH 3 - 6; time 5 - 8 h. B. Preparation of the culture medium: The sweet potato residue, bran, urea, and ammonium sulfate treated in step A are mixed in a certain proportion to obtain the culture medium. The mass ratio of the sweet potato residue to the bran is (2.5 - 3.5):1, the addition amount of urea is 3 - 5% of the total mass of the sweet potato residue and the bran, and the addition amount of ammonium sulfate is 3 - 5% of the total mass of the sweet potato residue and the bran. C, Fermentation: Add a multi-strain combination of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger to the culture medium for fermentation treatment; the addition amounts of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger are all 1-3% of the mass of the culture medium, and the concentrations of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger are all 1×10 8 cfu / mL, the fermentation time is 3-5 d, and the fermentation temperature is 25-35 °C.

2. The method for co-fermentation of fungal enzyme for sweet potato residue according to claim 1, characterized in that: In step A, the enzymatic hydrolysis combination and parameters are Amy 141.3 U / g, Cel 247.5 U / g, Xyl 27.1 U / g, temperature 46.5°C; pH 5.1; time 6.4 h.

3. The enzymatic and microbial co-fermentation method for sweet potato residues according to claim 1, characterized in that: In step B, the mass ratio of the sweet potato residue to the bran is 3:1, the addition amount of urea is 4% of the total mass of the sweet potato residue and the bran, and the addition amount of ammonium sulfate is 4% of the total mass of the sweet potato residue and the bran.

4. The method for co-fermentation of fungal enzyme for sweet potato residue according to claim 1, characterized in that: In step C, the addition amounts of Candida utilis, Lactobacillus plantarum JM113, Bacillus subtilis, and Aspergillus niger are all 2% of the mass of the culture medium, the fermentation time is 4 d, and the fermentation temperature is 31°C.

5. The enzymatic and microbial co-fermentation method for sweet potato residues according to claim 1, characterized in that: The preservation number of Lactobacillus plantarum JM113 is CCTCC NO: M2015680.

6. The method for co-fermentation of fungus and enzyme for sweet potato residues according to claim 1, characterized in that: The culturing method of the Lactobacillus plantarum JM113 comprises the following steps: using the chicken digestive tract contents and mucosa as the sample source, performing 10-fold serial dilutions until reaching 10 -7 , taking 200 μl from each dilution gradient and spreading it on the MRS solid medium, placing it in an incubator at 37 °C for inverted culture for 48 h, and preserving it after purification by picking colonies and streaking.

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