CN113832055B - Klebsiella grimontii and application of Klebsiella grimontii in preparation of protein beverage from fermented aquatic product - Google Patents

Abstract

The invention discloses Klebsiella grimontii and application thereof in preparing protein beverage from fermented aquatic products, and belongs to the technical field of microorganisms. The Klebsiella grimontii strain has a preservation number of CCTCC NO: klebsiella grimontii R4 of M2021976. The Klebsiella grimontii bacterium fermented aquatic product can remove fishy smell and generate attractive aroma with cheese aroma, nut aroma, baking aroma and flower aroma, and can be used for preparing protein beverage from the fermented aquatic product. Inoculating Klebsiella grimontii into a fish culture medium for fermentation, filtering, blending, degassing, homogenizing, canning and sterilizing at high temperature to obtain the aquatic product protein beverage.

Description

Klebsiella grimontii and application of Klebsiella grimontii in preparation of protein beverage from fermented aquatic product

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a novel Klebsiella grimontii strain and application thereof in preparing protein beverage from a fermented aquatic product.

Background

With the improvement of the living standard of people, the demand of protein is more and more vigorous. Aquatic product resources contain rich protein, and protein beverages prepared from the aquatic product resources are widely concerned. At present, the method for preparing the aquatic product protein beverage mostly adopts an enzymatic hydrolysis method, but the problems of unsatisfactory sensory quality, heavy fishy smell and unacceptable consumer exist generally.

Disclosure of Invention

The invention aims to provide a novel Klebsiella grimontii strain and application of the novel Klebsiella grimontii strain in preparing protein beverage by utilizing fermented aquatic products, and the prepared protein beverage has no fishy smell and has attractive fragrance of cheese fragrance, nut fragrance, baking fragrance and flower fragrance.

The purpose of the invention is realized by the following technical scheme:

the Klebsiella grimontii strain has a preservation number of CCTCC NO: klebsiella grimontii R4 of M2021976. The strain was deposited at the China center for type culture Collection (address: university of Wuhan, china) at 08/06/2021.

An application of Klebsiella grimontii in preparing protein beverage from fermented aquatic products.

A method of preparing a protein beverage by fermenting a water product using Klebsiella grimontii bacteria, comprising the steps of: inoculating Klebsiella grimontii into a fish culture medium for fermentation, filtering, blending, degassing, homogenizing, canning and sterilizing at high temperature to obtain the aquatic product protein beverage. The fish meat comprises grass carp, black carp, silver carp, bighead carp, megalobrama, mandarin fish, weever and the like, and can be one or more than two mixed fish meat according to a required proportion. The fermentation time is preferably 2-7d.

The fish culture medium comprises fish juice, glucose and NaCl. The fish meat culture medium is preferably prepared by a method comprising the following steps: adding water into fish meat, mincing, mixing, and filtering to obtain filtrate (fish juice); adding glucose and NaCl into the filtrate, and sterilizing to obtain the fish culture medium. Wherein, the mass ratio of the fish meat to the water is preferably 1-1; more preferably, the mass ratio of the fish meat to the water is 1.

The Klebsiella grimontii is preferably selected from the bacteria with the preservation number of CCTCC NO: m2021976 Klebsiella grimontii R4.

An aquatic product protein beverage is obtained by the method.

The invention has the beneficial effects that: the invention finds a new application from the obtained Klebsiella grimontii. Experiments prove that the screened Klebsiella grimontii can produce the fragrance of cheese, nut, baking, flower and the like when fermenting aquatic products, and can be used as a fermentation strain for preparing protein beverages by utilizing the aquatic products.

Drawings

FIG. 1 is a colony morphology of Klebsiella grimontii.

FIG. 2 is a GC-MS total ion flow diagram after inoculating Klebsiella grimontii bacteria in fish culture medium and fermenting and culturing for 2d at 37 ℃.

FIG. 3 is a GC-MS total ion flow diagram of fish culture medium inoculated with Klebsiella grimontii bacteria after fermentation culture at 37 ℃ for 7d.

FIG. 4 is a GC-MS total ion flow diagram of a fish culture medium after non-inoculated fermentation strain is subjected to fermentation culture at 37 ℃ for 2 days.

Detailed Description

The embodiments of the present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the reagent materials used in the following examples were purchased from conventional biochemical stores, unless otherwise specified.

The raw material for separating and purifying the strain is stinky mandarin fish purchased from Shannong food industry park, inc. in Hubei, which is stored at-18 ℃ and is transported to a laboratory for sampling after being purchased.

Self-made aroma-producing bacteria screening culture medium: chopping grass carp and fish meat, adding water according to a mass ratio of 1; adding glucose 2%, naCl 1%, and sterilizing at 115 deg.C for 20min to obtain the final product.

Example 1 isolation and purification of Klebsiella grimontii

Wiping different parts of a plurality of smelly mandarin fish with sterilized cotton swabs to obtain bacteria-containing liquid, diluting the bacteria-containing liquid with 0.9% normal saline to a proper concentration, coating the bacteria-containing liquid in an MRS agar plate by using a sterilized glass triangular rod, placing the plate in a constant-temperature incubator at 37 ℃, selecting a single colony after 24-72h, scribing and separating the single colony on the plate, and performing staining microscopy to detect the colony morphology and purifying the single colony. Inoculating the purified strain slant to a self-made aroma-producing strain screening culture medium by using an inoculating loop, and performing shake culture at constant temperature of 37 ℃ for 2d at 120 r/min. Odor screening finds that the fragrance generated by the fermentation of the three strains R4, R7 and R8 is pleasant and strong, wherein the fragrance of R4 is the strongest. 0.5mL of each of the 3 samples was placed in a 20mL headspace vial and incubated at 45 ℃ for 10min. Analysis was performed using an electronic nose model PEN3 from AIRSENSE, germany, headspace gas was injected into the sensor chamber at 150mL/min, measurement was started and continued for 150s. The electronic nose analysis result also shows that the fragrance generated by R4 fermentation has the maximum signal on W5S (nitrogen oxide compound), W1S (methyl compound) and W1W (sulfide) sensors. Therefore, R4 can be used as a potential fermentation strain for preparing protein beverage by using aquatic products.

And (3) conveying the slant of the R4 strain obtained after purification and screening to perform strain identification, wherein the strain identification is completed by Wuhan Tianyihui-Yuan biotechnology limited company. The R4 is identified to belong to Klebsiella grimontii, the strain is named as Klebsiella grimontii R4, and the strain is preserved in China Center for Type Culture Collection (CCTCC) (Wuhan university in Wuhan, china), the preservation date is 2021, 08 and 06 days, the preservation number is CCTCC NO: m2021976.

The colony morphology of Klebsiella grimontii R4 is shown in FIG. 1. The colony on the MRS culture medium plate is in a medium-sized circular shape, is white, has rough surface gloss, is provided with bulges and is opaque, and the edge is neat; gram-positive, spherical, oxidase-negative, catalase-positive, without motility.

In the following examples, the Klebsiella grimontii strain used is a strain with a collection number of CCTCC NO: m2021976, klebsiella grimontii R4.

Example 2 application of Klebsiella grimontii bacteria in preparation of protein beverage by fermenting grass carp

Grass carp (with weight of not less than 2 kg) purchased in a large-market aquatic product trading area of agricultural products in Baishazhou is transported to a laboratory with water, and is slaughtered, peeled and taken. Mixing fish meat with purified water according to a ratio of 1; adding 2% glucose and 1% NaCl by mass into the filtrate, and sterilizing at 115 deg.C for 20min to obtain fish culture medium.

Inoculating Klebsiella grimontii into a fish culture medium, culturing for 2d and 7d at constant temperature of 120r/min and 37 ℃, respectively, passing through 100-mesh gauze, removing larger particles in fermentation liquor, adding sucrose to adjust sugar content to 7%, degassing at a vacuum degree of 20-30kPa, homogenizing for 2 times by using a high-pressure homogenizer, canning, homogenizing under a pressure of 40MPa, and finally sterilizing the canned fermentation liquor in a high-pressure sterilization pot by using a sterilization formula of 10-15-10min/121 ℃ to obtain the grass carp fermented protein beverage which is yellowish in color, thick in texture, rich in cheese aroma, nut aroma, baking aroma and flower aroma.

Putting 4mL of grass carp fermented protein beverage into a 20mL sample bottle, adding 2-methyl-3-heptanone as an internal standard to enable the concentration of the internal standard to be 20.4 mu g/L, balancing the internal standard in a constant-temperature water bath at 40 ℃ for 20min, extracting the internal standard for 40min at constant temperature by using an aged CAR/PDMS extraction head, and inserting the extraction head into a GC-MS sample inlet to desorb the internal standard for 5min at 250 ℃ for aroma analysis. Chromatographic conditions are as follows: DB-WAX polar capillary column (30 m × 0.25mm,0.25 μm), he as carrier gas, flow rate of 1mL/min, no shunt sampling; adopting temperature programming: the initial temperature is 30 ℃, the temperature is kept for 5min, then the temperature is raised to 70 ℃ at 3 ℃/min, then the temperature is raised to 120 ℃ at 2.5 ℃/min, then the temperature is raised to 180 ℃ at 3 ℃/min, and finally the temperature is raised to 220 ℃ at 8 ℃/min, and the temperature is kept for 5min.

The GC-MS total ion flow graph of the fish culture medium inoculated with Klebsiella grimontii bacteria after fermentation culture at 37 ℃ for 2 days is shown in figure 2, the GC-MS total ion flow graph of the fish culture medium inoculated with Klebsiella grimontii bacteria after fermentation culture at 37 ℃ for 7 days is shown in figure 3, and the GC-MS total ion flow graph of the blank fish culture medium inoculated with no fermentation bacteria after fermentation culture at 37 ℃ for 2 days is shown in figure 4. The peak area ratios were used to calculate the concentrations of the aroma compounds, as shown in table 2.

TABLE 1 concentration of aroma compounds in grass carp protein beverages fermented by Klebsiella grimontii strain at different times

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Note: blank refers to a sample obtained after a blank fish culture medium which is not inoculated with a fermentation strain is subjected to fermentation culture at 37 ℃ for 2 d; the description of the fragrance of each compound refers to the handbook of edible spice compiled by Sunpo nationality.

As can be seen from the data in Table 1, after the Klebsiella grimontii bacteria ferment the fish culture medium for 2 days, the concentrations of the 2 fish-smell key aroma compounds, namely hexanal and nonanal, are remarkably changed, the hexanal completely disappears, and the nonanal content is remarkably reduced. At the same time, acetoin in cheese aroma, 3-ethyl-2, 5-dimethylpyrazine in nut aroma, baking aroma, octanol, nonanol, decanol and β -phenylethyl alcohol in fruit aroma, flower aroma are also produced. The grass carp protein beverage fermented for 2 days is endowed with cheese fragrance, nut fragrance, baking fragrance and flower fragrance. And continuing fermentation for 7 days, and generating a large amount of pyrazine compounds including methylpyrazine, 2, 5-dimethylpyrazine, 2, 3-dimethylpyrazine, 2-ethyl-6-methylpyrazine, 2,3, 5-trimethylpyrazine, 3-ethyl-2, 5-dimethylpyrazine, 2-ethyl-3, 5-dimethylpyrazine, 2,3,5, 6-tetramethylpyrazine, 2,3, 5-trimethyl-6-ethylpyrazine and the like, so that the grass carp protein beverage presents strong nut flavor and baking flavor. Therefore, the grass carp protein beverage can be endowed with the aroma of cheese aroma, nut aroma, baking aroma and flower aroma after combination to different degrees by controlling the fermentation time within 2 to 7 days.

Example 3 application of Klebsiella grimontii bacteria in preparing protein beverage by fermenting Mandarin fish

The same as example 2, except that: the mandarin fish (with the weight of more than or equal to 500 g) purchased from the large-market aquatic product trading area of the Baishazhou agricultural products is transported to a laboratory with water, and is slaughtered, peeled and taken out.

Example 4 use of Klebsiella grimontii bacteria for fermenting Wuchang fish for preparing protein beverages

The same as example 2, except that: transporting Wuchang fish (the weight of the fish is more than or equal to 500 g) purchased in the big market aquatic product trading area of the agricultural products of Baishazhou with water to a laboratory, slaughtering, peeling and taking meat.

TABLE 2 inoculation of Klebsiella grimontii bacteria for fermentation 2d to prepare the concentration of aroma compounds in Siniperca chuatsi and Wuchang fish protein beverages

As can be seen from the data in Table 2, after the Klebsiella grimontii bacteria ferment the fish culture medium of mandarin fish and Wuchang fish for 2 days, hexanal completely disappears, and the nonanal content is also obviously reduced. Meanwhile, the fish meat culture media of 2 kinds of fish all produced acetoin in cheese flavor, 2, 5-dimethylpyrazine, 2,3, 5-trimethylpyrazine, 3-ethyl-2, 5-dimethylpyrazine, 2-ethyl-3, 5-dimethylpyrazine and 2,3,5, 6-tetramethylpyrazine in nut flavor, roasted flavor, 2-methylpentanol, octanol, nonanol, decanol and β -phenylethyl alcohol in sweet flavor, flower flavor, and the like. Therefore, after fermentation treatment by Klebsiella grimontii, the fishy smell of the protein beverages prepared from different raw materials was reduced, and similar cheese, nut, roasted and flowery flavors were exhibited.

The above embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and the technical solution of the present invention is covered by the claims of the present invention.

Claims (8)

1. Klebsiella grignard, (B) and (C)Klebsiella grimontii) The method is characterized in that: for protection ofThe collection number is CCTCC NO: m2021976 Klebsiella grimontii R4.

2. Use of the klebsiella pneumoniae of claim 1 in the preparation of a protein beverage by fermenting a water product.

3. A method for preparing a protein beverage by fermenting a marine product with klebsiella pneumoniae as set forth in claim 1, which comprises: the method comprises the following steps: inoculating the Klebsiella pneumoniae of claim 1 into a fish culture medium for fermentation, and filtering, blending, degassing, homogenizing, canning and sterilizing at high temperature to obtain the aquatic product protein beverage; the fish culture medium contains fish juice, glucose and NaCl.

4. The method of claim 3, wherein: the fish meat comprises grass carp, black carp, silver carp, bighead carp, megalobrama amblycephala, mandarin fish and weever.

5. The method of claim 3, wherein: the fermentation time is 2-7 days.

6. The method of claim 3, wherein: the fish culture medium is prepared by the method comprising the following steps: adding water into fish meat, mincing, mixing, and filtering to obtain filtrate; adding glucose and NaCl into the filtrate, and sterilizing to obtain fish culture medium.

7. The method of claim 6, wherein: the mass ratio of the fish meat to the water is 1-1.

8. An aquatic product protein beverage is characterized in that: obtained by the process of any one of claims 3 to 7.

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