Fermentation Process For Preparing Postbiotic With By-Product Of Wheat Processing

Abstract

A fermentation process for preparing a postbiotic with a by-product of wheat processing includes: preparation of a material; inoculation of Paenibacillus polymyxa (P. polymyxa)+Brachybacterium paraconglomeratum (B. paraconglomeratum)+Flavobacterium pectinovorum (F. pectinovorum) as fermentation strains; and isolation and extraction of the postbiotic. Since wheat starch wastewater and wheat bran include abundant nutrient components, the wheat starch wastewater and wheat bran can be used to prepare postbiotics, which is conducive to reducing a wastewater treatment cost of a related enterprise and protecting the environment, and can turn waste into treasure and increase an added value of an agricultural product. Preliminary experimental results show that, when no nutrients are additionally supplemented, marker products such as lipoteichoic acid (LTA) and short-chain fatty acids (SCFAs) in a prepared postbiotic solution are at high contents, which can extend an industrial chain of wheat deep-processing and improve an industrial value of deep processing technology.

Claims

1. A fermentation process for preparing a postbiotic with a by-product of wheat processing, comprising the following steps: (1) preparation of a material thoroughly mixing a process water and a wheat bran resulting from wheat starch processing in a mass ratio of (1-12):1 to obtain a resulting mixture, sterilizing the resulting mixture to obtain a sterilized mixture, and cooling the sterilized mixture to room temperature to obtain a fermentation matrix; (2) inoculation of fermentation strains preparing each of Paenibacillus polymyxa (P. polymyxa), Brachybacterium paraconglomeratum (B. paraconglomeratum), and Flavobacterium pectinovorum (F. pectinovorum) into a seed culture, and mixing resulting seed cultures to obtain a mixed fermentation broth; and under sterile conditions, inoculating the mixed fermentation broth into the fermentation matrix in step (1) according to a mass proportion of 1% to 25%, and conducting fermentation at 25? ? C. to 37? C. for 48 h to 192 h; and (3) isolation and extraction of the postbiotic after the fermentation in step (2) is completed, filtering a resulting fermentation system to obtain a resulting filtrate, subjecting the resulting filtrate to primary centrifugation to obtain a first supernatant, and collecting the first supernatant; and subjecting the first supernatant to secondary centrifugation to obtain a second supernatant, and collecting the second supernatant obtained after the secondary centrifugation to obtain a postbiotic solution, wherein main components of the postbiotic comprise a lipoteichoic acid (LTA) and a short-chain fatty acid (SCFA).

2. The fermentation process for preparing the postbiotic with the by-product of wheat processing according to claim 1, wherein in step (1), the process water and the wheat bran resulting from wheat starch processing are mixed in a mass ratio of (8-10):1.

3. The fermentation process for preparing the postbiotic with the by-product of wheat processing according to claim 1, wherein in step (2), the fermentation strains are as follows: a P. polymyxa strain with an accession No. of CGMCC 1.15984; a B. paraconglomeratum strain with an accession No. of CGMCC 1.838; and an F. pectinovorum strain with an accession No. of CGMCC 1.12362.

4. The fermentation process for preparing the postbiotic with the by-product of wheat processing according to claim 1, wherein in step (2), an inoculation proportion is 10%.

5. The fermentation process for preparing the postbiotic with the by-product of wheat processing according to claim 1, wherein in step (2), a number of viable bacteria in the seed culture prepared with each of the P. polymyxa, the B. paraconglomeratum, and the F. pectinovorum is greater than or equal to 1?10.sup.6/mL; and when the mixed fermentation broth is prepared, the seed cultures are in a volume ratio of 1:1:1.

6. The fermentation process for preparing the postbiotic with the by-product of wheat processing according to claim 1, wherein in step (3), the primary centrifugation is conducted at 1,000 r/min for 30 min; and the secondary centrifugation is conducted at 5,000 r/min for 30 min.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows an impact of different bacterial strain combinations on a fermentation effect; and

[0023] FIG. 2 shows fermentation effects of mixed bacteria of various bacterial strains in different ratios.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The technical solutions of the present disclosure are further described below with reference to embodiments. Before specific embodiments are introduced, in order to facilitate those skilled in the art to understand the relevant research and development in the present disclosure in detail, some background experiments in the following embodiments are briefly described as follows.

Biological Materials:

[0025] Inoculated strains used during fermentation: P. polymyxa deposited in CGMCC on Nov. 30, 2016 with the accession No. CGMCC 1.15984, B. paraconglomeratum deposited in CGMCC with the accession No. CGMCC 1.838, and F. pectinovorum deposited in CGMCC on Oct. 15, 2012 with the accession No. CGMCC 1.12362, which are publicly-available strains and all are purchased directly from the China General Microbiological Culture Collection Center (CGMCC).

[0026] P. polymyxa refers to a group of rod-like bacteria with diversified physiological characteristics, which are spore-forming gram-positive bacteria; P. polymyxa lives in an aerobic or facultatively-anaerobic environment; P. polymyxa can decompose starches or polysaccharides, can produce acids, and can also produce active substances with functions of antagonizing microorganisms, promoting plant growth, or the like; and P. polymyxa may be Polymyxin, Colistin, Circulin, Jolipeptin, Polypeptins, Gatavalin, or Fusaricidins, for example.

[0027] B. paraconglomeratum refers to a group of club-shaped gram-positive bacteria, which can produce amylases.

[0028] F. pectinovorum can convert starches into organic acids (such as SCFAs) and other active ingredients during fermentation.

[0029] It should be explained and emphasized that the microbial strains that can produce amylases or starches in the prior art are not limited to the above three strains, but given the similar growth conditions of the above three strains (optimal growth temperature: 25? ? C. to 37? C., and optimal growth pH: about 7.0) and based on the convenience of unified control of growth conditions and the technical purpose of preparation of a postbiotic through fermentation in the present application, the above three strains are comprehensively selected for a specific fermentation experiment.

By-Products of Wheat Processing:

[0030] A process water sample and a wheat bran sample resulting from wheat starch processing are all from Henan Feitian Agricultural Development Co., Ltd.; and main nutrients and contents thereof in the samples are as follows:

TABLE-US-00001 TABLE 1 Starch water composition (%, mass percentage) Total sugar, Crude protein, Crude fat, Starch, Crude fiber, Moisture content, Item % % % % % % Starch 1.6 0.7 0 0.1 0.6 97.0 water

TABLE-US-00002 TABLE 2 Wheat bran composition (%, mass percentage) Item Total sugar Crude protein Crude fat Starch Crude fiber Crude ash Wheat bran (dry basis) 39.63 18.66 2.28 27.6 6.92 4.91

EXAMPLES

[0031] With specific by-products of wheat processing as an example, the fermentation process for preparing a postbiotic with a by-product of wheat processing designed in the present application is specifically described by the inventors as follows:

(1) Preparation of a Material

[0032] The process water and wheat bran resulting from wheat starch processing were thoroughly mixed in a mass ratio of 5:1, sterilized at 121? C. for 20 min, and then cooled to room temperature to obtain a fermentation matrix.

(2) Inoculation of Fermentation Strains

[0033] Each of P. polymyxa (CGMCC 1.15984, strain A), B. paraconglomeratum (CGMCC 1.838, strain B), and F. pectinovorum (CGMCC 1.12362, strain C) was prepared into a seed culture in which a number of viable bacteria was 1?10.sup.6, and resulting seed cultures were mixed according to a volume ratio of 1:1:1 to obtain a mixed fermentation broth in which a total number of viable bacteria was 1?10.sup.6; and [0034] under sterile conditions, the mixed fermentation broth was inoculated into the fermentation matrix in step (1) according to a mass proportion of 10%, and fermentation was conducted at 28? C. for 192 h.

(3) Isolation and Extraction of the Postbiotic (Index Components: LTA and SCFA)

[0035] After the fermentation in step (2) was completed, a resulting fermentation system was filtered, a resulting filtrate was subjected to primary centrifugation at 1,000 r/min for 30 min, and a resulting supernatant was collected; and [0036] the collected supernatant was subjected to secondary centrifugation at 5,000 r/min for 30 min, a supernatant obtained after the secondary centrifugation was collected to obtain a postbiotic solution, and the postbiotic solution was stored at 4? C.

[0037] From the perspective of increasing a content of a postbiotic active ingredient, in order to determine an appropriate fermentation process, relevant fermentation process conditions were specifically explored by the inventors, and a specific process was briefly described as follows:

[0038] It should be noted that an LTA content was used as an evaluation index for an effect of the postbiotic (the LTA content was detected by an LTA ELISA kit of Shanghai Lanpai Biotechnology Co., Ltd. with reference to instructions of the kit), and SCFA was used as a marker component of the postbiotic to evaluate an effect of the postbiotic in the postbiotic solution (an SCFA content was detected by an SCFA ELISA kit of Shanghai Win-Win biochemical Co., Ltd. with reference to instructions of the kit).

(A) Impact of a Single Strain or a Mixed Strain on LTA and SCFA in a Fermentation Broth

[0039] In order to investigate fermentation effects of single-strain fermentation and multi-strain fermentation, the strain A, the strain B, the strain C, and combinations of different strains (when different strains were used in combination, the different strains were in a volume ratio of 1:1 or 1:1:1; and before inoculation, a number of bacteria was 1?10.sup.6, that is, a number of viable bacteria was 1?10.sup.6 when a single strain was inoculated, and an initial total number of viable bacteria was also 1?10.sup.6 when a combination of different strains was inoculated) each were inoculated for fermentation.

[0040] Fermentation was conducted at 28? ? C. for 192 h, and LTA and SCFA contents in a resulting fermentation broth were detected. Specific results were shown in FIG. 1.

[0041] It can be seen from FIG. 1 that, during either single-strain fermentation or multi-strain fermentation, the LTA and SCFA contents gradually increase with the extension of a fermentation time, but after a specified fermentation time, the LTA and SCFA contents are basically stable or even decrease, and thus a fermentation time of 45 h to 100 h can be adopted from the perspective of actual production efficiency; and from the perspective of an actual fermentation yield, the multi-strain fermentation leads to a better fermentation effect than the single-strain fermentation, where the fermentation with the strains A, B, and C has the optimal fermentation effect. Therefore, without increasing a production cost, it is most suitable to use a mixture of the three strains for fermentation. Therefore, subsequently, a mixture of the three strains is used for fermentation to prepare a postbiotic.

[0042] Specifically, in terms of a fermentation yield of a single strain, an SCFA yield of the strain A is 116.8, an SCFA yield of the strain B is 93.4, and an SCFA yield of the strain C is 64.8, indicating that the strain A has obvious advantages; and an LTA yield of the strain B was 255.0, an LTA yield of the strain A was 210.4, and an LTA yield of the strain C was 178.0, indicating that, although the strain B has the highest LTA yield, the strain A also has obvious advantages.

[0043] In terms of active substance yields after fermentation with mixed bacteria, fermentation effects after fermentation with mixed bacteria in different ratios are shown in FIG. 2. With reference to comparison among the inoculation proportions in FIG. 1, it can be seen that a fermentation effect is significantly improved when different strains are used in a ratio of 1:1.

(B) Impact of a Fermentation Time on a Fermentation Effect of Mixed Bacteria

[0044] Since it was determined that the fermentation with the three strains had the optimal fermentation effect, in order to further determine an impact of a fermentation time, with reference to the above operations, a fermentation time in step (3) was adjusted (the three strains were mixed in a ratio of 1:1:1) to investigate an impact of a fermentation time on a fermentation effect. Specific results were shown in Table 3 below.

TABLE-US-00003 TABLE 3 Impacts of different fermentation times of multi-strain fermentation on LTA and SCFA contents in a fermentation broth Fermentation time, h 0 12 24 48 96 192 LTA content (pg/mL) 122.5 183.6? 258.9? 318.4? 318.8 318.6 Improvement rate, % 49.88 41.01 22.98 0.13? ?0.06? SCFA content (?g/mL) 0 68.9? 89.6? 116.4? 116.8 116.9 Improvement rate, % 30.04 29.91 0.34? 0.09? Note: ? or ? in the table indicates an increase or a decrease.

[0045] The improvement rate is calculated as follows:

[00001]$R=\frac{C_n-C_0}{C_0}?100\%,$ [0046] where C.sub.0 represents a concentration (?g/mL) of LTA or SCFA detected at a stage before fermentation, C.sub.n represents a concentration (?g/mL) of LTA or SCFA detected at a stage after continuous fermentation, and R represents an improvement rate (%).

[0047] It can be seen from Table 3 that the LTA and SCFA contents gradually increase with the extension of a fermentation time, but are basically stable after 48 h of fermentation, indicating that 48 h is the optimal fermentation time; and an actual fermentation time can be 45 h to 50 h.

(C) Impact of a Material Ratio on a Fermentation Effect

[0048] Since the optimal fermentation bacterial solution ratio and the optimal fermentation time were determined in steps (A) and (B), respectively, in order to further investigate an impact of a material ratio on a fermentation effect, with reference to the above operations, a material ratio in step (1) was adjusted (the fermentation time in step (2) was 48 h, and the three strains were mixed according to a ratio of 1:1:1) to investigate an impact of a material ratio on a fermentation effect. Specific results were shown in Table 4 below.

TABLE-US-00004 TABLE 4 Impacts of different process water-to-wheat bran ratios on LTA and SCFA contents in a fermentation broth (fermentation time: 48 h) Process water-to-wheat bran ratio 1:1 3:1 5:1 8:1 10:1 15:1 LTA content (pg/mL) 122.6 130.5? 194.4? 318.6? 354.9? 303.9? Improvement rate, % 6.44 48.97 63.89 11.39? ?14.37? SCFA content (?g/mL) 0 68.1? 88.5? 115.1? 115.4 115.5 Improvement rate, % 29.96 30.06 0.26? 0.09?

[0049] It can be seen from Table 4 that the LTA and SCFA contents gradually increase with the increase of the process water-to-wheat bran ratio, but decrease slightly when the process water-to-wheat bran ratio exceeds 15:1, and thus the optimal process water-to-wheat bran ratio is 8:1 to 10:1.

(D) Impact of an Inoculum Size on a Fermentation Effect

[0050] Since an inoculum size during fermentation also often bad a specified impact on a fermentation effect, with reference to the above operations, the inventors adjusted an inoculum size in step (2) (a material ratio in step (1) was 8:1 and a fermentation time in step (2) was 48 h) to investigate an impact of an inoculum size on a fermentation effect. Specific results were shown in Table 5 below.

TABLE-US-00005 TABLE 5 Impacts of different inoculum sizes on LTA and SCFA contents in a fermentation broth (fermentation time: 48 h) Inoculum size, % 1 5 10 15 20 25 LTA Content 122.6 130.5? 318.6? 346.1? 415.3? 498.4? (pg/mL) Improvement 6.44 144.14 8.63 19.99 20.01 rate, % SCFA Content 0 70.1? 91.1? 118.5? 128.8? 141.9? (?g/mL) Improvement 29.96 30.08 8.69? 10.17? rate, %

[0051] It can be seen from Table 5 that the LTA content gradually increases with the increase of the inoculum size, but the SCFA content is basically stable after the inoculum size exceeds 20%, and thus the optimal inoculum size is 10% to 15%.

INDUSTRIAL APPLICABILITY

[0052] With the present disclosure, nutrients in wheat starch wastewater and wheat bran can be deeply extracted to prepare a postbiotic solution including high contents of LTA and SCFA without additionally supplementing nutrients. The present disclosure is conducive to reducing a wastewater treatment cost of a related enterprise and protecting the environment, can turn waste into treasure and increase an added value of an agricultural product, and can also lay a specified technical foundation for extending an industrial chain of wheat deep-processing and improving an industrial value of deep processing technology.

[0053] Finally, it should be noted that the above embodiments are merely intended to illustrate the technical solutions of the present disclosure, rather than to limit the present disclosure. Those of ordinary skill in the art may make other modifications or equivalent replacements to the technical solutions of the present disclosure without departing from the spirit and scope of the technical solutions of the present disclosure, but such modifications or equivalent replacements should fall within the scope defined by the claims of the present disclosure.