Method for preparing functional konjac oligosaccharide by using pectobacterium carotovorum

Abstract

The invention discloses a method for preparing functional konjac oligosaccharide by using pectobacterium carotovorum, which is characterized in that pectobacterium carotovorum is separated and purified from konjac tubers with soft rot phenomenon, and is used for hydrolyzing konjac glucomannan after being subjected to enlarged culture, and the functional konjac oligosaccharide has a good hydrolysis effect on the konjac glucomannan, so that the functional konjac oligosaccharide can be prepared by using the pectobacterium carotovorum. The obtained konjac oligosaccharide cannot be degraded into monosaccharide in stomach and intestinal tracts, but can promote functional lactic acid bacteria to generate a certain amount of lactic acid, and has a very good functional effect, which has very good theoretical value and practical significance for improving the processing utilization rate and additional value of konjac.

Description

Technical Field

[0001] The invention belongs to the technical field of konjac deep processing, and particularly relates to a method for preparing functional konjac oligosaccharides by using carrot soft rot pectin bacteria. Background Art

[0002] Konjac is a special food resource with health-care effects. Konjac tofu, which is solidified by boiling alkali into tofu-like shape, is the main processed food. Konjac is rich in glucomannan, which accounts for more than 45% of dried konjac. Konjac glucomannan has a variety of physiological activities that are beneficial to health, such as anti-obesity and anti-inflammatory effects, so it is widely used in the field of functional foods and is used in food additives because of its non-toxic and harmless characteristics. However, due to its large molecular weight, konjac glucomannan swells into a colloid when it comes into contact with water, making it difficult to swallow. In addition, konjac glucomannan is difficult to be further digested, absorbed and utilized in the human gastrointestinal tract, so it cannot be used directly. Except for being used as a food thickener, it is difficult to add a large amount of it to processed foods as an additive, and its dosage and functional role are limited. Compared with konjac glucomannan, moderate hydrolysis of konjac glucomannan into konjac oligosaccharides without affecting its functional effects can increase the solubility of glucomannan and improve its fluidity after absorbing water. After entering the human intestine, it can be used by probiotics to promote its growth and improve the structure of human intestinal flora. It is a highly potential prebiotic and is increasingly valued in the food and pharmaceutical industries. Traditional enzymes for degrading glucomannan mostly use β-mannanase or endoglucanase, but the result of their action is to completely hydrolyze glucomannan and lose the function of glucomannan. Summary of the invention

[0003] The purpose of the present invention is to provide a method for preparing functional konjac oligosaccharides by using carrot soft rot pectin bacteria, which not only preserves the functionality of konjac polysaccharides, but also increases the solubility and fluidity after water absorption.

[0004] The method for preparing functional konjac oligosaccharides using Pectinobacterium carotovora provided by the present invention comprises the following steps:

[0005] Step 1: Isolation of Pectobacterium carotovora from konjac tubers infected with soft rot

[0006] Samples were randomly selected from the rotten area of ​​konjac tubers infected by soft rot, and the samples were mixed with sterile water to make a suspension, which was inoculated on konjac culture medium for cultivation; in each sample, a culture medium with a transparent liquid and a circular transparent area on the surface was selected, and the liquid in the circular transparent area was dipped with an inoculation loop and re-inoculated on LB modified culture medium, and cultured at 28-32°C until visible colonies appeared on the surface of the culture medium; the strains of different colonies obtained were inoculated into the screening culture medium, and cultured at 28-32°C for 22-26h; the strains that could grow and produce on the screening culture medium were selected For the strains with obvious bacterial clusters and significantly reduced viscosity of the culture medium, the selected strains were inoculated into LB liquid culture medium under a sterile environment, cultured at 28-32°C for 11-13h, centrifuged at 5000-6000r / min for 9-11min, and the obtained precipitate was washed 2-3 times with sterile water to obtain pure mycelium; the mycelium was placed in a PE tube, pre-cooled at -20--15°C, and then vacuum-freeze-dried to finally obtain freeze-dried carrot soft-rot pectin bacteria, which was refrigerated at -20--15°C for later use.

[0007] Step 2: Expand the culture of Pectinobacterium spp.

[0008] The preserved carrot soft rot pectinobacterium strains were activated with LB liquid medium, and the LB liquid medium was used for shaking and expanding culture at 28-32°C and a shaking speed of 160-200 r / min for 18-24 h to obtain a concentration of 1×10 6 ~1×10 8 / mL of carrot soft rot pectinobacterium suspension.

[0009] Step 3: Hydrolyze konjac flour

[0010] Whey protein, yeast extract and water are added to konjac flour, and the mixture is fully mixed and swollen to prepare a viscous konjac flour fermentation liquid, and the pH value thereof is adjusted to 7.1-7.5 with NaOH; the konjac flour fermentation liquid is added to a triangular flask, and the mixture is kept warm at 95-100 DEG C for 18-22 minutes, and after cooling, a suspension of carrot soft rot pectin bacillus is inoculated under aseptic conditions with an inoculation amount of 9%-10%, the mouth of the triangular flask is sealed, and the mixture is oscillated and hydrolyzed at 40-43 DEG C and a shaking table speed of 110-130 r / min for 18-24 hours; after the hydrolysis is completed, the mixture is heated and kept warm at 75-85 DEG C for 13-17 minutes for sterilization.

[0011] Step 4: Isolation of Konjac Oligosaccharides

[0012] The sterilized hydrolyzate is dialyzed using a 2000Da dialysis bag for 20 to 24 hours, and then centrifuged at 2000 to 4000 r / min for 15 to 20 minutes. The hydrolyzate is dried using a spray dryer or a vacuum freeze dryer to obtain powdered konjac oligosaccharides.

[0013] In the above step 1, the composition of the konjac culture medium is: 9-11 g / L konjac powder, natural pH, and sterilized at 121° C. for 18-22 min.

[0014] In the above step 1, the composition of the LB modified medium is: 9-11 g / L tryptone, 4.5-5.5 g / L yeast extract, 9-11 g / L NaCl, 4.5-5.5 g / L konjac flour, pH is adjusted to 7.5, and sterilized at 121° C. for 18-22 min.

[0015] In the above step 1, the composition of the screening culture medium is: 9-11 g / L konjac flour, 9-11 g / L yeast extract, 3.5-4.5 mL / L glycerol, 0.8-1.2 g / L K2HPO4 and KH2PO4, the pH is adjusted to 7.0-8.0, and sterilized at 121°C for 18-22 min.

[0016] In the above step 3, the konjac flour fermentation liquid preferably contains 16.00-18.60 g / L of konjac flour, 7.0-9.0 g / L of whey protein, and 9.0-11.0 g / L of yeast extract.

[0017] In the above step 4, the obtained konjac oligosaccharide consists of oligosaccharides with a degree of polymerization of 2 to 9 and no free hemiacetal hydroxymannose, wherein the oligosaccharide contains non-reducing terminal glucose and non-reducing terminal mannose, but no reducing terminal glucose, and the disaccharide structure is mannose-mannose and glucose-mannose.

[0018] The beneficial effects of the present invention are as follows:

[0019] The invention separates and purifies carrot soft rot pectin bacteria from konjac tubers with soft rot phenomenon, and uses the bacteria for hydrolyzing konjac glucomannan after expanded culture, showing good hydrolysis effect on konjac glucomannan. The obtained konjac oligosaccharides cannot be degraded into monosaccharides in the stomach and intestines, but can promote functional lactic acid bacteria to produce a certain amount of lactic acid, and have good functional effects. This method has good theoretical value and practical significance for improving konjac processing utilization rate and added value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The growth morphology of the isolated strain in LB modified medium (a) and the morphology after Gram staining (10×40) (b).

[0021] Figure 2is the phylogenetic tree of strains.

[0022] Figure 3 The effect of hydrolysis time on the hydrolysis rate of konjac glucomannan and the yield of konjac oligosaccharides.

[0023] Figure 4 This is the matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis spectrum of konjac glucomannan hydrolysate.

[0024] Figure 5 It is the hydrolysis rate of konjac oligosaccharide in simulated gastric fluid (pH=1).

[0025] Figure 6 is the hydrolysis rate of konjac oligosaccharides in simulated intestinal fluid.

[0026] Figure 7 It is the effect of konjac oligosaccharides on the acid production of Lactobacillus. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0028] Konjac soft rot is a disease that is very easy to occur in the planting process of konjac, and the soft rot phenomenon occurs due to the rot of the rhizome. The present invention separates and purifies carrot soft rot pectin bacteria from the konjac tubers with the soft rot phenomenon, and hydrolyzes the konjac after expanded culture, and investigates the influence of the hydrolysis conditions on the hydrolysis effect of konjac, and the specific test is as follows:

[0029] 1. Isolation and expansion of Pectinobacterium carotovora from konjac tubers infected with soft rot

[0030] Samples were randomly selected from the rotten areas of konjac tubers affected by soft rot, and the samples were mixed with sterile water to make a suspension, which was inoculated on konjac culture medium for cultivation. In each sample, a culture medium with a transparent liquid on the surface and a circular transparent area was selected, and the circular transparent area liquid was dipped with an inoculation loop and re-inoculated on LB modified culture medium, and the plate was placed under 30°C until visible colonies appeared on the surface of the culture medium. The composition of the konjac culture medium is: 10g / L konjac powder, natural pH, sterilized at 121°C for 20min. The composition of the LB modified culture medium is: 10g / L tryptone, 5.0g / L yeast extract, 10g / L NaCl, 5g / L konjac powder, pH adjusted to 7.5, sterilized at 121°C for 20min.

[0031] The strains of different colonies obtained above were inoculated into a 150 mL triangular flask of screening medium and cultured at 30 ° C for 24 h. Strains that can grow on the screening medium, produce obvious bacterial clusters and significantly reduce the viscosity of the medium were selected. The composition of the screening medium is: 10 g / L konjac flour, 10 g / L yeast extract, 4 mL / L glycerol, 1.0 g / L K2HPO4 and KH2PO4, pH is adjusted to 7.5, and sterilized at 121 ° C for 20 min.

[0032] The selected strains were inoculated into LB liquid medium under a sterile environment, cultured at 30°C for 12h, centrifuged at 6000r / min for 10min, and the obtained precipitate was washed 3 times with sterile water to obtain pure bacterial cells. The bacterial cells were placed in a PE tube, pre-cooled at -20°C, and then vacuum-freeze-dried to finally obtain isolated strains, which were refrigerated at -20°C for standby use. The composition of the LB liquid medium is: no konjac flour is added, and the rest is the same as the LB modified medium.

[0033] Use an optical microscope at a magnification of 40× to observe the Gram staining of the isolated strains and their growth on LB modified medium, and take photos and record the results. Figure 1 As shown. Figure 1 Observation under an optical microscope revealed that the isolated strain was white, opaque, and had a smooth surface. After 12 hours of culture, it grew well on LB modified medium. The surface was smooth with slight ridges. The colonies were irregular in shape and had a rotten, foul smell. They were long-chain, rod-shaped, and Gram-negative bacteria. The isolated strain was sent to a biological company for gene extraction, PCR amplification, and sequencing. After obtaining the sequencing data, the sequence alignment analysis was performed using the GenBank database of NCBI. Based on the results obtained, the MEGA 10.2.6 software was used to construct a phylogenetic tree based on specific parameters and models. The results are shown in Figure 2. Figure 2 As shown. Figure 2 It can be seen that the isolated strain is most closely related to the 16S rDNA of Pectobacterium carotovorum Geo_Ac.1.22 in terms of gene sequence (homology>99.99%). The strain was further identified as Pectobacterium carotovorum.

[0034] The preserved carrot soft rot pectin bacteria strains were activated with LB liquid medium, and the LB liquid medium was used for agitation expansion culture at 30°C and a shaking table speed of 180 r / min for 18 to 24 h to make the bacterial suspension concentration reach 1×10 6 ~1×10 8 / mL.

[0035] 2. Optimization of the hydrolysis process of konjac glucomannan

[0036] After adding konjac flour to distilled water, stir it continuously to make it fully swell without agglomeration, and then sterilize the swollen konjac glucomannan (KGM) colloid at 120℃ for 20min. Set the KGM substrate concentration to 16g / L, the hydrolysis pH to 7, and the hydrolysis time to 24h. The effects of hydrolysis temperatures of 30℃, 35℃, 40℃, 45℃, and 50℃ on the hydrolysis rate of KGM colloid were explored. The results showed that the optimal hydrolysis temperature was 40℃. At the optimal hydrolysis temperature of 40℃, the hydrolysis time was set to 24h, and the effects of pH of 5, 6, 7, 8, and 9 on the hydrolysis rate of KGM colloid were explored. The results showed that the optimal pH was 6. Select the optimal pH condition and the hydrolysis time to be 24h. Explore the effects of KGM concentrations of 10g / L, 13g / L, 16g / L, 19g / L, and 22g / L on the hydrolysis rate of KGM colloid. The results showed that 16g / L was the optimal KGM colloid concentration.

[0037] On the basis of single factor experiment, whey protein, yeast extract and water were added to KGM colloid according to the ratio of 8.0g / L whey protein and 10.0g / L food-grade yeast extract, and the mixture was fully mixed and swollen to make a viscous konjac flour fermentation liquid, and its pH was adjusted with NaOH. After sterilization, the carrot soft rot pectin bacteria were inoculated with an inoculation amount of 10%, and the shaking table speed was 120r / min for 24h. The hydrolysis effect of carrot soft rot pectin bacteria on konjac glucomannan was determined. With hydrolysis temperature, pH, KGM colloid concentration as factors and hydrolysis effect as an indicator, the Box-Behnken central composite experimental design principle was used to optimize the optimal process through response surface analysis, and the influence of different independent variables on the hydrolysis effect value was compared. The independent variable factor level is shown in Table 1, and the experimental results are shown in Table 2. Hydrolysis effect value = oligosaccharide yield × 50% + hydroxyl radical scavenging rate × 50%. The method for determining the hydroxyl radical scavenging rate is as follows: 1 mL of 5 mmol / mL H2O2 aqueous solution, 1 mL of 6 mmol / mL FeSO4 aqueous solution, and 1 mL of 20 mmol / mL salicylic acid ethanol solution are added to 1 mL of hydrolyzate in sequence, shaken well, and reacted at 37°C for 40 minutes. After the reaction is completed, the absorbance value A is measured at 510 nm. i The following formula is the calculation formula for the scavenging rate of oligosaccharides on hydroxyl radicals:

[0038]

[0039] Where: A0 is the absorbance at 510nm after 1mL of distilled water replaces the hydrolyzate; A j It is the absorbance value at 510nm after 1mL of distilled water replaces the FeSO4 aqueous solution.

[0040] Table 1 Levels of independent variables in the response surface analysis experimental design

[0041]

[0042] Table 2 Experimental results of response surface quadratic model

[0043]

[0044] Through regression fitting, the corresponding binary multiple regression equation is obtained: hydrolysis effect value (%) = 51.58 + 2.19A + 0.6531B + 0.0169C-0.7175AB + 1.58AC-0.5337BC-7.67A 2 -1.41B 2 -2.41C 2 .

[0045] The parameter optimization analysis of the regression equation model established by the response surface test results showed that the optimal process conditions for hydrolyzing konjac glucomannan by carrot soft rot pectin bacteria were: hydrolysis temperature of 41.36℃, pH of 7.27, and KGM colloid concentration of 16.82g / L. The theoretical value of the hydrolysis effect was 51.79%. Considering the operability of actual food industry production, the specific test values ​​were adjusted, and three parallel tests were carried out under the conditions of hydrolysis temperature of 41℃, pH=7.3, and KGM colloid concentration of 16.80g / L. The hydrolysis effect value was 50.68%.

[0046] 3. Effect of hydrolysis time on the hydrolysis of konjac glucomannan by Pectobacterium carotovora

[0047] Based on the above experiments, the time of hydrolysis of konjac glucomannan by Pectinobacterium carotovora was studied, and the changes of hydrolysis rate and oligosaccharide yield of konjac glucomannan hydrolysate at different times were determined. Figure 3 .

[0048] from Figure 3 It can be seen that as the time of the action of carrot soft rot pectin bacteria on konjac glucomannan increases, the hydrolysis rate of glucomannan and the yield of oligosaccharides gradually increase, but after the time is extended to 20h, the increase rate tends to be gentle. From 20h to 24h, the hydrolysis rate of glucomannan increases from 37.63% to 40.21%, and the yield of oligosaccharides increases from 42.54% to 44.10%, and the increase is not large. The experiment selected 20h of hydrolysis as the appropriate action time.

[0049] Example 1

[0050] 1. Isolation of Pectobacterium carotovora from konjac tubers infected with soft rot

[0051] Samples were randomly selected from the rotten area of ​​konjac tubers infected by soft rot, mixed with sterile water to make a suspension, and inoculated on konjac culture medium for culture. In each sample, a culture medium with transparent liquid and a circular transparent area on the surface was selected, and the liquid in the circular transparent area was dipped with an inoculation loop and re-inoculated on LB modified culture medium. The plate was placed at 30°C until visible colonies appeared on the surface of the culture medium.

[0052] The strains of different colonies obtained above were inoculated into a 150mL triangular flask of screening medium and cultured at 30°C for 24h. Select strains that can grow on the screening medium, produce obvious bacterial clusters and significantly reduce the viscosity of the medium. Inoculate the selected strains into LB liquid culture medium under a sterile environment, culture at 30°C for 12h, centrifuge at 6000r / min for 10min, and wash the obtained precipitate with sterile water 3 times to obtain pure bacteria. Put the bacteria into a PE tube, pre-cool it at -20°C, and then freeze-dry it in a vacuum to finally obtain carrot soft rot pectin bacteria, which are refrigerated at -20°C for use.

[0053] 2. Expanded culture of carrot soft rot pectin bacteria

[0054] The preserved carrot soft rot pectinobacterium strains were activated with LB liquid medium, and then cultured with LB liquid medium at 30°C and a shaking table speed of 180 r / min for 24 h to obtain a concentration of 1×10 6 ~1×10 8 / mL of carrot soft rot pectinobacterium suspension.

[0055] 3. Hydrolysis of konjac flour

[0056] By the proportioning of konjac powder 16.80g / L, lactalbumin 8.0g / L and food grade yeast extract 10.0g / L, in konjac powder, add lactalbumin, yeast extract and water, fully mix swelling, make viscosity konjac powder fermented liquid, and it is 7.3 to regulate its pH with NaOH.Konjac powder fermented liquid is added in triangular flask, 100 ℃ of insulation 20min, inoculate carrot soft rot pectin bacillus suspension under aseptic condition after cooling, inoculation size 10% (v / v), seal triangular flask mouth, oscillate hydrolysis 24h under 41 ℃, shaking table speed 120r / min condition.After hydrolysis finishes, 80 ℃ of heating insulation 15min, carry out sterilization treatment.

[0057] 4. Separation and purification of konjac oligosaccharides

[0058] The sterilized hydrolyzate was dialyzed using a 2000Da dialysis bag for 24 hours, and then centrifuged at 3000r / min for 20 minutes. After freeze-drying, konjac oligosaccharide was obtained, which was recorded as KGO-1.

[0059] The composition of the hydrolyzate KGO-1 was analyzed by thin layer chromatography and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry was performed in positive ion reflectron mode with a laser power density of 106 W / cm 2 The accelerating potential was 5 kV. The results were analyzed using FlexAnalysis 3.4 software. Figure 4 .from Figure 4 It can be seen that the m / z value of the KGO-1 ion, a hydrolyzate of konjac glucomannan by Pectinobacterium carotovora, is between 437.2454 and 1337.3921, with a degree of polymerization of 2 to 9. The ion intensity between the mass-to-charge ratio values ​​less than 437.2454 and greater than 1337.3921 is extremely low, indicating that the ion intensity content within this range is low. In general, according to the signal height of matrix-assisted laser desorption time-of-flight mass spectrometry, the relative content of oligosaccharide products gradually decreases with the increase of degree of polymerization, and the intensity of oligomers with a degree of polymerization of 2 is less than 5×10 3 From the figure, we can see that the degree of polymerization of the main product is 3, and the possible products in the trimer ion cluster are mainly natural trimers ([M+Na] + = m / z 527.54) and the native trimer oligomer with signal acetyl groups ([M+Na+acetyl] + = m / z 569.53. Further analysis by nuclear magnetic resonance spectroscopy showed that the hydrolysis of konjac glucomannan by Pectinobacterium carotovora could produce monoacetylated, diacetylated and triacetylated natural oligosaccharides with a degree of polymerization of 3 to 9.

[0060] In summary, konjac glucomannan was completely degraded into oligosaccharides after hydrolysis by Bacillus carotovora. The color did not change significantly before and after hydrolysis. KGO-1 maintained a white appearance and had a more significant flaky network structure. No new glycosidic bonds were generated before and after hydrolysis. The main structure of KGM did not change due to the hydrolysis by Bacillus carotovora or β-mannanase. -1 There is a characteristic peak of acetyl at the end, which is a natural oligosaccharide with an acetyl modification; and the hydrolysis process does not affect the pyranose ring structure of konjac glucomannan. The oligosaccharide structure is stable before and after hydrolysis, and the oligosaccharide does not contain protein and nucleic acid. The prepared KGO-1 is an oligosaccharide composition with a degree of polymerization of 2 to 9 and does not contain free hemiacetal hydroxymannose, which has non-reducing terminal glucose and non-reducing terminal mannose, but not reducing terminal glucose, and the disaccharide structure is mannose-mannose and glucose-mannose.

[0061] The above KGO-1 was used to conduct digestion experiments and probiotic function experiments. The specific experimental methods and results are as follows:

[0062] (1) In vitro digestion experiment of hydrolysate

[0063] ①Simulated gastric digestion experiment

[0064] According to the composition of the digestive juice in the stomach, the digestive juice was prepared in vitro for the experiment. First, 10 mL of the sample solution treated with simulated oral digestive juice was mixed with the gastric digestive juice, and the in vitro simulated gastric digestion experiment was carried out for different times. After the digestion was completed, the enzyme was inactivated in a 100°C water bath for 5 minutes to terminate the reaction. Centrifuge at 4°C and 8000r / min for 15 minutes, remove the precipitate, and store in a -20°C refrigerator. The digestion results of oligofructose, KGO-1 and konjac glucomannan (KGM) in simulated gastric juice in vitro are shown in Figure 5 .

[0065] Depend on Figure 5 It can be seen that when the pH value of the simulated gastric fluid environment is 1, after 6 hours of hydrolysis, the hydrolysis rate of oligofructose increased from 13.31% to 30.10%, with the highest degree of hydrolysis, indicating that oligofructose was hydrolyzed in the simulated gastric fluid; the hydrolysis rate of KGO-1 did not change significantly, with a hydrolysis rate of 5.11%, and the hydrolysis rate of KGM was higher than that of KGO-1.

[0066] ②Simulated small intestinal digestion experiment

[0067] According to the composition of the digestive juice in the small intestine, the small intestinal digestive juice was prepared for the experiment. First, 20 mL of the sample solution treated with simulated gastric digestive juice was mixed with the small intestinal digestive juice, and the in vitro simulated small intestinal digestion experiment was carried out for different times. After the digestion was completed, the enzyme was inactivated in a 100°C water bath for 5 minutes to terminate the reaction. Centrifuge at 4°C and 8000r / min for 15 minutes, remove the precipitate, and store in a -20°C refrigerator. The digestion results of oligofructose, KGO-1 and konjac glucomannan (KGM) in vitro simulated small intestinal juice are shown in Figure 6 .

[0068] like Figure 6 As shown in the figure, the hydrolysis rates of konjac glucomannan and KGO-1 did not change significantly during digestion in simulated intestinal fluid, indicating that the three components have good anti-hydrolysis properties during digestion in small intestinal fluid and will not be hydrolyzed in simulated intestinal fluid.

[0069] (2) Effect of konjac oligosaccharides on the proliferation of functional lactic acid bacteria

[0070] Figure 7This is an experiment on the effect of konjac oligosaccharides on lactobacillus acid production. The acid production effects of Lactobacillus paracasei (Figure a) and Lactobacillus plantarum (Figure b) in the culture medium containing glucose, oligofructose, and KGO-1 are different. Both strains of Lactobacillus can use konjac oligosaccharides to ferment and produce lactic acid, and their contents increase with the extension of fermentation time, but are lower than glucose and oligofructose. Glucose and oligofructose as carbon sources promote lactobacillus lactic acid production better than konjac oligosaccharides. KGO-1 can improve the ability to produce lactic acid compared to konjac glucomannan. Among them, KGO-1 has the best prebiotic effect. When fermented for 24 hours, KGO-1 promotes the production of lactic acid in Lactobacillus paracasei and Lactobacillus plantarum to 5.84mg / mL and 4.84mg / mL respectively; Lactobacillus paracasei has the best lactic acid production ability. When fermented for 24 hours, KGO-1 promotes the production of lactic acid in Lactobacillus paracasei and Lactobacillus plantarum to 5.84mg / mL and 5.18mg / mL respectively.

[0071] Combination Figure 6 and Figure 7 The experimental results showed that carrot soft-rot pectin bacteria hydrolyzed konjac glucomannan, and the oligosaccharide KGO-1 produced could not be degraded into monosaccharides in the stomach and intestines, but could promote the functional lactic acid bacteria to produce a certain amount of lactic acid. Further measurement of the bacteria in the fermentation broth showed that the concentration (optical density) of the bacteria continued to increase. All these confirmed that konjac oligosaccharides have good functionality.

Claims

1. A method for preparing functional konjac oligosaccharides using Pectinobacterium carotovora, characterized in that: The method comprises the following steps: Step 1: Isolation of Pectobacterium carotovora from konjac tubers infected with soft rot Samples were randomly selected from the rotten area of ​​konjac tubers infected by soft rot, and the samples were mixed with sterile water to make a suspension, which was inoculated on konjac culture medium for cultivation; in each sample, a culture medium with a transparent liquid and a circular transparent area on the surface was selected, and the liquid in the circular transparent area was dipped with an inoculation loop and re-inoculated on LB modified culture medium, and cultured at 28-32°C until visible colonies appeared on the surface of the culture medium; the strains of different colonies obtained were inoculated into the screening culture medium, and cultured at 28-32°C for 22-26h; the strains that could grow and produce on the screening culture medium were selected For strains with obvious bacterial clusters and significantly reduced culture medium viscosity, the selected strains were inoculated into LB liquid culture medium under a sterile environment, cultured at 28-32°C for 11-13 hours, centrifuged at 5000-6000 r / min for 9-11 minutes, and the obtained precipitate was washed 2-3 times with sterile water to obtain pure mycelium; the mycelium was placed in a PE tube, pre-cooled at -20--15°C, and then vacuum-freeze-dried to finally obtain freeze-dried carrot soft rot pectin bacteria, which was refrigerated at -20--15°C for later use; Step 2: Expand the culture of Pectinobacterium spp. The preserved carrot soft rot pectinobacterium strains were activated with LB liquid medium, and the LB liquid medium was used for shaking and expanding culture at 28-32°C and a shaking speed of 160-200 r / min for 18-24 h to obtain a concentration of 1×10 6 ~1×10 8 / mL of carrot soft rot pectinobacterium suspension; Step 3: Hydrolyze konjac flour whey protein, yeast extract and water are added to konjac flour, the mixture is fully mixed and swollen to prepare a viscous konjac flour fermented liquid, and the pH value thereof is adjusted to 7.1-7.5 with NaOH; the konjac flour fermented liquid is added to a triangular flask, and the mixture is kept warm at 95-100° C. for 18-22 minutes, and after cooling, a suspension of carrot soft rot pectin bacillus is inoculated under aseptic conditions with an inoculation amount of 9%-10%, the mouth of the triangular flask is sealed, and the mixture is oscillated and hydrolyzed at 40-43° C. and a shaking table speed of 110-130 r / min for 18-24 hours; after the hydrolysis is completed, the mixture is heated and kept warm at 75-85° C. for 13-17 minutes to perform a sterilization treatment; Step 4: Isolation of Konjac Oligosaccharides The sterilized hydrolyzate is dialyzed using a 2000Da dialysis bag for 20 to 24 hours, and then centrifuged at 2000 to 4000 r / min for 15 to 20 minutes. The hydrolyzate is dried using a spray dryer or a vacuum freeze dryer to obtain powdered konjac oligosaccharides.

2. The method for preparing functional konjac oligosaccharides using Pectinobacterium carotovora according to claim 1, characterized in that: In step 1, the composition of the konjac culture medium is: 9-11 g / L konjac powder, natural pH, and sterilized at 121° C. for 18-22 min.

3. The method for preparing functional konjac oligosaccharides using Pectinobacterium carotovora according to claim 1, characterized in that: In step 1, the composition of the LB improved medium is: 9-11 g / L tryptone, 4.5-5.5 g / L yeast extract, 9-11 g / L NaCl, 4.5-5.5 g / L konjac flour, pH is adjusted to 7.5, and sterilized at 121° C. for 18-22 min.

4. The method for preparing functional konjac oligosaccharides using Pectinobacterium carotovora according to claim 1, characterized in that: In step 1, the screening culture medium is composed of: 9-11 g / L konjac flour, 9-11 g / L yeast extract, 3.5-4.5 mL / L glycerol, 0.8-1.2 g / L K2HPO4 and KH2PO4, the pH is adjusted to 7.0-8.0, and sterilized at 121° C. for 18-22 min.

5. The method for preparing functional konjac oligosaccharides using Pectinobacterium carotovora according to claim 1, characterized in that: In step 3, the konjac flour fermentation liquid contains 16.00-18.60 g / L of konjac flour, 7.0-9.0 g / L of whey protein, and 9.0-11.0 g / L of yeast extract.

6. The method for preparing functional konjac oligosaccharides using Pectinobacterium carotovora according to claim 1, characterized in that: In step 4, the obtained konjac oligosaccharide consists of oligosaccharides with a degree of polymerization of 2 to 9 and no free hemiacetal hydroxymannose, wherein the oligosaccharide contains non-reducing terminal glucose and non-reducing terminal mannose, but no reducing terminal glucose, and the disaccharide structures are mannose-mannose and glucose-mannose.

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