Fermented shrimp paste and bacterial strain used by same

Abstract

The invention discloses fermented shrimp paste and a bacterial strain used by the same. The strain for fermenting the shrimp paste is at least one of aspergillus, aspergillus niger, mucor and / or rhizopus oryzae. The fermented shrimp paste has excellent indexes such as amino nitrogen, volatile basic nitrogen, flavor, color and the like.

Description

Technical Field

[0001] The invention relates to the field of microbial food, in particular to a fermented shrimp paste and a strain used therefor. Background Art

[0002] Black tiger shrimp, also known as giant tiger shrimp, has a breeding output of 128,420 tons in China in 2023, and 35-45% of shrimp heads and shells will be produced as by-products during processing. The heads of black tiger shrimp are larger than those of white shrimp and contain more soluble components, and are considered to be a high-quality shrimp by-product. Shrimp by-products contain a variety of nutrients such as unsaturated fatty acids, carotenoids, free amino acids, etc., which can be used to prepare chitin, lipids, astaxanthin and condiments. The heads of white shrimp can be prepared into low-salt shrimp paste by adding koji prepared by Aspergillus oryzae. Due to the low protein content of shrimp shells, previous studies have shown that the shells of white shrimp added with soybeans can be fermented with Aspergillus oryzae to obtain shrimp paste with an amino acid nitrogen content of 1.13-1.44g / 100g. Studies have shown that shrimp shells and shrimp heads can be fermented into shrimp paste for high-value utilization.

[0003] Shrimp paste is a traditional aquatic condiment made by grinding low-value shrimps such as grasshopper shrimp and hair shrimp after adding salt and fermentation, but the production cycle of traditional shrimp paste is long, and the quality and flavor are unstable. Aspergillus niger, Rhizopus oryzae and Mucor are safe strains certified by the US Food and Drug Administration and can be used in the production of fermented foods. Aspergillus niger, Rhizopus oryzae and Mucor are also widely used in traditional fermented soy products in China, and they play an important role in accelerating the degradation of raw materials and improving the flavor of products. Aspergillus niger can promote the degradation of protein in the brewing process of soy sauce to reduce the bitterness of the product, Rhizopus oryzae can promote the fermentation of wheat bran to produce a variety of aromatic components to improve the smell, and Mucor can promote the production of small molecular flavor peptides and amino acids in the fermentation process of fermented bean curd to enhance the umami taste of the product. In the application of aquatic condiments, the fermentation of kelp and soybeans with Aspergillus oryzae and Aspergillus niger is conducive to the formation of flavor substances such as valeraldehyde, phenylethanol and 2-octenal in kelp sauce. However, there is no research on the use of Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger and Mucor for fermenting shrimp paste on the market. Summary of the invention

[0004] The invention aims to provide a shrimp paste with better nutrition, taste and flavor.

[0005] To achieve the above object, the present invention provides a use of Aspergillus niger, Aspergillus niger, Mucor and / or Rhizopus oryzae for preparing fermented shrimp paste.

[0006] The present invention also provides a fermented shrimp paste, characterized in that the strain used for fermenting the shrimp paste is at least one of Aspergillus, Aspergillus niger, Mucor and / or Rhizopus oryzae.

[0007] Furthermore, when it is a mixed strain of Mucor, Aspergillus oryzae and Rhizopus oryzae, the weight ratio of the three is: 3:1:3; 3:3:2; 2:3:3; 1:3:3; 1:1:1; 3:2:1; 2:3:1; 1:2:2; or 2:1:2.

[0008] Further, when it is a single strain, the preparation method of fermented shrimp paste is as follows: weigh soybeans, soak them in water for more than 10 hours, drain them, steam them together with shrimp shells and shrimp heads, and then crush them with a meat grinder to obtain a mixture of soybeans, shrimp shells and shrimp heads; wherein the mass ratio of soybeans: shrimp shells: shrimp heads is 2:1:1; the mixture is inoculated with the strain, and then koji is made in a constant temperature and humidity chamber; the koji making temperature is 28°C, the humidity is 90%, and the koji making time is 48 hours; then, brine containing 10% sodium chloride is added to the obtained koji according to the mass ratio of koji: brine of 1:2, and after mixing evenly, it is transferred to a fermentation jar, fermented in a 50°C constant temperature chamber for 28 days, inactivated enzymes in a water bath at 90°C for 30 minutes, and then filtered with a 10-40 mesh sieve to remove the coarse shrimp shells to obtain a shrimp paste sample;

[0009] When it is a mixed strain, the preparation method of fermented shrimp paste is as follows: each strain is inoculated at a mass percentage of 0.125% into a mixture of cooled cooked shrimp heads, shrimp shells and soybeans, and koji is made in a constant temperature and humidity chamber at a temperature of 28°C and a humidity of 90% for 48 hours; then, brine containing 10% sodium chloride is added to the obtained koji at a mass ratio of 1:2 between koji and brine, and after uniform mixing, the mixture is transferred to a fermentation jar, fermented in a constant temperature chamber at 50°C for 28 days, inactivated in a water bath at 90°C for 30 minutes, and then filtered through a 10-40 mesh sieve to remove the coarse shrimp shells to obtain a shrimp paste sample.

[0010] The embodiment of the present invention verifies that fermented shrimp paste prepared with Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger and Mucor has good nutritional ingredients, flavor and taste, and sensory organs. The fermented shrimp paste prepared with Aspergillus oryzae, Rhizopus oryzae and Mucor in different proportions is further compared. Taking various factors into consideration, it is found that when the weight ratio of Mucor, Aspergillus oryzae and Rhizopus oryzae is: 3:1:3; 3:3:2; 2:3:3; 1:3:3; 1:1:1; 3:2:1; 2:3:1; 1:2:2; or 2:1:2, the total quality of the fermented shrimp paste is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a graph showing the effect of bacterial strains on amino nitrogen and volatile basic nitrogen in shrimp paste.

[0012] Figure 2 This is a diagram showing the effect of bacterial strains on the apparent morphology of fermented shrimp paste.

[0013] Figure 3 It is a 3D topographic map and two-dimensional difference map of shrimp paste fermented by different strains.

[0014] Figure 4 It is the fingerprint spectrum of volatile compounds in shrimp paste fermented by different strains.

[0015] Figure 5 This is the clustering heat map of ROAV values ​​of volatile compounds in shrimp paste fermented by different strains.

[0016] Figure 6 This is the result chart of volatile compounds in shrimp paste fermented with multiple strains of bacteria.

[0017] Figure 7 This is the fingerprint result of volatile compounds in shrimp paste fermented with multiple strains of bacteria. DETAILED DESCRIPTION

[0018] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0019] In the following embodiments:

[0020] Shrimp heads and shells: Fujian Shengjiang Food Technology Co., Ltd.;

[0021] Soybean: Fujian Fangwei Food Technology Co., Ltd.;

[0022] Aspergillus oryzae: Yishui Jinrun Biotechnology Co., Ltd.;

[0023] Rhizopus oryzae, Aspergillus niger, and Rhizopus oryzae: Shandong Hezhong Kangyuan Biotechnology Co., Ltd.;

[0024] Salt: China National Salt Industry Co., Ltd.

[0025] Phthalic acid 9-fluorenylmethyl chloroformate: Agilent, USA; other reagents were of analytical grade.

[0026] YS3010 spectrophotometer: Shenzhen Sanenshi Technology Co., Ltd.; 1200 high performance liquid chromatograph: Agilent, USA; TS-5000Z electronic tongue system: Insent, Japan; 5K1-100039 gas chromatography-ion mobility spectrometer: Shandong Haineng Scientific Instrument Co., Ltd.; 877 potentiometric titrator: Metrohm, Switzerland.

[0027] Example 1: Preparation of shrimp paste

[0028] Preparation of shrimp paste koji: Weigh 2kg of soybeans, soak them in water for 12 hours, drain them, steam them with 1kg of shrimp shells and 1kg of shrimp heads for 30 minutes, and crush them with a meat grinder to obtain a mixture of soybeans, shrimp shells and shrimp heads. Inoculate the mixture with 1.25g of Aspergillus oryzae, Aspergillus niger, Mucor and Rhizopus oryzae, respectively, and then make koji in a constant temperature and humidity chamber. The koji making temperature is 28°C, the humidity is 90%, and the koji making time is 48 hours.

[0029] Preparation of shrimp paste: Add salt water containing 10% sodium chloride to the shrimp paste koji obtained above according to the mass ratio of koji: salt water 1:2, mix well and transfer to a fermentation jar, ferment in a 50°C constant temperature box for 28 days, and take samples every 7 days. After the fermentation, the shrimp paste is inactivated in a water bath at 90°C for 30 minutes, and then filtered through a 10-mesh sieve to remove the coarse shrimp shells to obtain the shrimp paste sample, which is stored at -18°C for subsequent experiments.

[0030] Embodiment 2: the performance measurement of shrimp paste:

[0031] Shrimp paste: prepared as in Example 1.

[0032] 1. Determination of amino nitrogen content: Determine according to the formaldehyde titration method reported by Cao Jiayan et al. (Cao Jiayan, Shi Linfan, Ren Zhongyang, et al. Effect of salt concentration on physicochemical properties and flavor substances during shrimp paste fermentation [J]. Journal of Chinese Institute of Food Science and Technology, 2024). Accurately weigh 5.0g shrimp paste, dissolve it in 100mL distilled water and filter it with 0.45μm filter paper. Take 20mL of the filtrate and mix it with 60mL distilled water, and then measure it with a potentiometric titrator. Calculate the amino nitrogen content of shrimp paste according to formula (1).

[0033]

[0034] Where: V1 is the volume of sodium hydroxide standard titration solution consumed after formaldehyde is added to the sample diluent for determination (mL); V2 is the volume of sodium hydroxide standard titration solution consumed after formaldehyde is added to the reagent blank test (mL); 0.014 is the mass of nitrogen equivalent to 1mL of sodium hydroxide standard solution (1mol / L) (g); m is the mass of the sample (g).

[0035] 2. Determination of volatile basic nitrogen: According to the method of Huang Li et al. (Huang Li, Shi Linfan, Ren Zhongyang, et al. Effect of electrolytic water pretreatment on quality changes of refrigerated large yellow croaker [J]. Journal of Fisheries of China, 2022, 46(07): 1210-1219), accurately weigh about 5g of shrimp paste and dissolve it in 50mL of distilled water and soak it for 30min, then filter it with 0.45μm filter paper. The filtrate was determined by microdiffusion method, where the insulation temperature was 37℃ and the time was 2h, and the volatile basic nitrogen content of shrimp paste was calculated according to formula (2).

[0036]

[0037] Wherein, V1 is the volume of hydrochloric acid solution consumed by the sample (mL); V2 is the volume of hydrochloric acid solution consumed by the reagent blank (mL); c is the concentration of the hydrochloric acid standard solution (mol / L); m is the mass of shrimp paste (g).

[0038] Results: See Figure 1 As can be seen from (a), the amino nitrogen content of various shrimp pastes showed an upward trend with the extension of fermentation time. At any fermentation time, the amino nitrogen content of shrimp paste prepared by Mucor fermentation was the highest, followed by shrimp paste prepared by Rhizopus oryzae, Aspergillus oryzae and Aspergillus niger fermentation. After 28 days of fermentation with Mucor, the amino nitrogen content of shrimp paste was 0.79g / 100g, which was lower than that of Huang Ailian et al. (Huang Ailian, An Min, Luo Meiyan, et al. Study on the process of preparing shrimp paste by rapid fermentation of white shrimp heads and koji [J]. Chinese Condiments, 2023, 48(08): 106-110) The amino nitrogen content of shrimp head paste obtained by fermenting shrimp heads prepared by Aspergillus oryzae 3.042 for 48 hours was 0.93g / 100g. Generally, the highly active protease secreted by Mucor can degrade the protein in fermented fermented bean curd into small molecules such as amino acids, while the enzyme system secreted by Aspergillus oryzae is mainly neutral and alkaline proteases. In the present application, the fermentation of the soybean paste is mainly carried out in a slightly acidic environment with a pH range of 6.09-6.42, which will quickly inactivate the alkaline protease secreted by Aspergillus oryzae, resulting in a decrease in the utilization rate of the protein in the soybean paste raw material.

[0039] from Figure 1(b) shows that with the extension of fermentation time, the volatile basic nitrogen content of shrimp paste fermented with Mucor first slowly increases and then tends to be stable, and the volatile basic nitrogen content of shrimp paste fermented with Aspergillus oryzae and Aspergillus niger both show a steady upward trend. However, the volatile basic nitrogen content of shrimp paste fermented with Rhizopus oryzae shows a rapid upward trend. At any fermentation time, the volatile basic nitrogen content of shrimp paste fermented with Mucor is the lowest, followed by Aspergillus niger, Rhizopus oryzae and Aspergillus oryzae. The volatile basic nitrogen content of the shrimp paste prepared in this study was 114-165 mg / 100 g, which was lower than the shrimp paste prepared by traditional fermentation by Gao et al. (GAO R, XUE J, SHI T, et al. Effects of'Bask in Sunlight and Dewed at Night' on the formation offermented flavor in Shrimp paste after maturation[J]. Food Chemistry, 2024, 452: 139546) (212 mg / 100 g). Figure 1 The results showed that among the shrimp paste fermented with four strains of bacteria, the shrimp paste prepared by fermentation with Mucor and Rhizopus oryzae not only had a high content of amino nitrogen but also produced a low content of volatile basic nitrogen, and was of better quality. The second best quality shrimp paste was that prepared with Aspergillus oryzae and Aspergillus niger.

[0040] 3. Color measurement: Use YS-3010 spectrophotometer to measure the color of shrimp paste. YS-3010 spectrophotometer is based on the standard printing plate (L * =91.86, a * =-0.88, b * =1.42) as the background to measure the brightness value (L * ), redness value (a * ), yellowness value (b * ).

[0041] The results are shown in Table 1 and Figure 2 .from Figure 2 It can be seen that the apparent colors of shrimp paste fermented by Aspergillus niger, Rhizopus oryzae, Aspergillus oryzae and Mucor are dark brown, yellow brown, yellow brown and red brown, respectively. * The value was the smallest, followed by shrimp paste fermented with Rhizopus oryzae, Aspergillus oryzae and Mucor. A similar trend also appeared in the a value of shrimp paste. * Value and b *The results showed that the shrimp paste prepared by Mucor fermentation had a good color. The color difference of shrimp paste fermented by Mucor, Aspergillus oryzae, Rhizopus oryzae and Aspergillus niger may be related to the degradation of carotene protein in the shrimp shell and the release of astaxanthin during the fermentation process, as well as the degree of Maillard reaction. Studies have found that during the fermentation process, carotene protein in the shrimp shell may be induced by protease to degrade to form free carotenoids, giving the shrimp paste a red color. The release of astaxanthin will cause the shrimp paste to appear red-orange, and the formation of Maillard reaction products will make the shrimp paste brown (PONGSETKUL J, BENJAKUL S, VONGKAMJAN K, et al. Changes in volatile compounds, Atp-related compounds and antioxidant properties of kapi, produced from acetes vulgaris, during processing and fermentation [J]. Food Bioscience, 2017, 19: 49-56). Heo et al. (HEO S, PARK J, LEE K, et al. Quality characteristics of soybean fermented by mucor, rhizopus, and aspergillus from meju[J]. Heliyon, 2023, 9(3)) found that the different colors of spores produced by Mucor, Rhizopus, and Aspergillus can lead to differences in the quality of fermented soybean. * Value, a * Value and b * Therefore, the color of fermented shrimp paste is not only related to the raw materials and the Maillard reaction of the fermentation process, but also affected by the strains.

[0042] Table 1 Effect of bacterial strains on shrimp paste color

[0043] Bacteria L* a* b* Aspergillus niger <![CDATA[22.68±0.21 d ]]> <![CDATA[2.72±0.02 d ]]> <![CDATA[3.69±0.03 d ]]> Rhizopus oryzae <![CDATA[28.93±0.06 c ]]> <![CDATA[10.84±0.02 c ]]> <![CDATA[18.35±0.08 c ]]> Aspergillus oryzae <![CDATA[35.08±0.04 b ]]> <![CDATA[16.03±0.14 b ]]> <![CDATA[26.88±0.37 b ]]> Mucor <![CDATA[35.51±0.05 a ]]> <![CDATA[18.25±0.03 a ]]> <![CDATA[31.19±0.05 a ]]>

[0044] Note: Different lowercase letters in the same column indicate significant differences (P<0.05)

[0045] 4. Determination of free amino acids: Determine according to the method reported by Sun Mengying et al. (Sun Mengying, Shi Linfan, Ren Zhongyang, et al. Nutritional evaluation and in vitro simulated digestion characteristics of European eel muscle protein [J]. Journal of Chinese Institute of Food Science and Technology, 2022, 22(06): 315-322). Accurately weigh 1g of shrimp paste and dissolve it in ultrapure water. Centrifuge and take the supernatant, add an equal volume of 10% (w / v) sulfosalicylic acid solution, and stand in the dark for 2 hours. Centrifuge and take the supernatant, condense to dryness at 55°C, then dissolve it with 0.02mol / L hydrochloric acid solution and make up to 5mL, and filter it with a 0.22μm water system membrane. The filtrate is derivatized with o-phthalaldehyde and 9-fluorenylmethyl chloroformate solution before column analysis by high performance liquid chromatography.

[0046] The results are shown in Table 2. Among the flavor amino acids of the four fermented shrimp pastes, glutamic acid had the highest content, and bitter amino acids accounted for the highest proportion. There was no significant difference in the total free amino acid content of shrimp pastes fermented with Mucor and Rhizopus oryzae, both of which were higher than those prepared by fermentation with Aspergillus oryzae and Aspergillus niger. The shrimp pastes fermented with Mucor and Rhizopus oryzae had a stronger umami taste, a high proportion of sweet amino acids, and a low proportion of bitter amino acids. The results of the study showed that the shrimp pastes fermented with Mucor and Rhizopus oryzae performed well in terms of amino acid flavor components.

[0047] Table 2 Effect of strains on free amino acid content in fermented shrimp paste (mg / 100g)

[0048]

[0049] Note: Umami amino acids: aspartic acid and glutamic acid;

[0050] Sweet amino acids: threonine, serine, proline, glycine, and alanine;

[0051] Bitter amino acids: valine, methionine, leucine, isoleucine, tyrosine, phenylalanine, histidine, lysine, and arginine;

[0052] Amino acid ratio = amino acid content / total free amino acid content.

[0053] Different lowercase letters in the same row indicate significant differences (P<0.05).

[0054] 5. Electronic tongue measurement: According to the method reported by Huang Li et al. (Huang Li, Shi Linfan, Ren Zhongyang, et al. Effect of electrolyzed water pretreatment on quality changes of refrigerated large yellow croaker [J]. Journal of Fisheries of China, 2022, 46(07): 1210-1219), 75 g shrimp paste was accurately weighed and mixed with 300 mL of distilled water, and then extracted at room temperature for 30 min. After filtering with 0.45 μm filter paper, the filtrate was used to measure the bitterness, bitter aftertaste, astringency, astringent aftertaste, sourness, umami, saltiness and richness of the shrimp paste taste using an electronic tongue.

[0055] The taste of shrimp paste fermented by different strains was analyzed by electronic tongue, and the results are shown in Table 3. Among the four fermented shrimp pastes, the shrimp paste fermented by Mucor has the highest umami and richness, and the lowest astringency and astringent aftertaste; followed by Rhizopus oryzae, and then Aspergillus oryzae and Aspergillus niger. Studies have found that the content and type of free amino acids directly affect the taste characteristics of shrimp paste, and umami amino acids are the main contributors to the umami taste of shrimp paste (HU M, ZHAO L, SUN H, et al. Recent advances on shrimp paste: key flavor components and biochemical formation pathways, biogenic amine formation, microbial functions, and innovative process strategies [J]. Trends in Food Science & Technology, 2024, 152: 104694). The results show that the shrimp paste prepared by fermentation of Mucor and Rhizopus oryzae is superior to the shrimp paste prepared by fermentation of Aspergillus oryzae and Aspergillus niger in taste.

[0056] Table 3 Effect of strains on the taste of fermented shrimp paste

[0057]

[0058] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0059] 6. Determination of volatile compounds: Referring to the method reported by Huang et al. (HUANG J, LIU Y, SHANG S, et al. Changes in bacterial flora and flavor of shrimp paste under different salt concentrations [J]. LWT, 2024, 205: 116534), the volatile compounds of shrimp paste were determined using gas chromatography-ion migration spectrometry (GC-IMS) equipped with an HP-5 chromatographic column. Accurately weigh 5.0 g of shrimp paste and place it in a 20 mL headspace bottle. After incubation at 80 ° C for 30 min, the sample was injected through the headspace. The specific procedure of IMS is to use 40 ° C as the initial temperature and maintain it for 5 min, then increase the temperature to 120 ° C at 10 ° C / min, then increase the temperature to 210 ° C at 20 ° C / min, and finally increase the temperature to 230 ° C at 2 ° C / min. The chromatographic analysis time is 30 minutes, the ionization source is a tritium source, the migration tube temperature is 45°C, and the drift gas is high-purity nitrogen (purity ≥ 99.999%). The NIST database and IMS database built into the Vocal software supporting GC-IMS are used to conduct qualitative analysis of the volatile compounds in shrimp paste, and the Reporter plug-in and Gallery Plot plug-in built into the software are used to draw a 3D topographic map, a two-dimensional difference map, and a fingerprint map of the volatile compounds in shrimp paste.

[0060] 1) 3D topographic maps and 2D difference maps of volatile compounds

[0061] In order to explore the differences in the types and contents of volatile compounds in shrimp paste fermented with different strains, GC-IMS was used to analyze shrimp paste fermented with Aspergillus oryzae, Aspergillus niger, Rhizopus oryzae and Mucor for 28 days. Figure 3 In a), the X, Y and Z axes represent the ion migration time, GC retention time and signal peak intensity, respectively. The red signal and the blue signal represent the high and low concentrations of volatile compounds, respectively. In the black frame, the concentration of volatile compounds in the shrimp paste fermented by Aspergillus niger is significantly higher than that in the shrimp paste fermented by Rhizopus oryzae, Aspergillus oryzae and Mucor. In the green frame, the signal peak distributions in the shrimp paste fermented by Aspergillus niger, Aspergillus oryzae, Rhizopus oryzae and Mucor are significantly different. In order to better reflect the differences in the types and contents of volatile compounds among the shrimp paste samples, the ion migration time and the reaction ion peak position of the 3D topographic map are normalized, and the shrimp paste fermented by Aspergillus niger is used as a reference to analyze and obtain a two-dimensional difference map between the shrimp paste samples ( Figure 3b). Blue spots indicate that the concentration of volatile compounds in the sample is lower than that in the reference sample, while white spots indicate that the concentration of volatile compounds in the sample is the same as that in the reference sample, and red spots indicate that the concentration of volatile compounds in the sample is higher than that in the reference sample. Moreover, the ion migration time of volatile compounds in shrimp paste fermented by different strains is concentrated in 1.0-2.0 ms, and the retention time is concentrated in 200-1200 s. In the black area, the types and contents of volatile compounds in shrimp paste fermented by Rhizopus oryzae, Aspergillus oryzae and Mucor are similar, and the concentrations of most compounds are lower than those in shrimp paste fermented by Aspergillus niger. In the green area, the concentrations of most volatile compounds are from high to low in the order of Aspergillus oryzae, Mucor and Rhizopus oryzae, but they are all higher than those in shrimp paste fermented by Aspergillus niger.

[0062] 2) Volatile compound fingerprint

[0063] In order to explore the effect of bacterial strains on the volatile compounds in fermented shrimp paste, the ion migration time and retention index were used for qualitative analysis, and the fingerprint of volatile compounds in shrimp paste was generated using the Gallery Plot plug-in ( Figure 4 ). The color of the spots in the figure indicates that the sample concentration is from low to high, from light to dark. Due to the different concentrations of volatile compounds, monomers or dimers may be produced, which are represented by (M) and (D), respectively. In area 1, the concentration of volatile compounds in shrimp paste fermented by Mucor is the lowest, including propyl butyrate, allyl isothiocyanate, 2-furfural (M) and isopropyl isothiocyanate. In area 2, the concentration of volatile compounds in shrimp paste fermented by Aspergillus niger is the highest, including undecane, 2-furfural (D), 3-methyl-2-butenal and 2-methylvaleraldehyde. In area 3, the concentration of volatile compounds in shrimp paste fermented by Rhizopus oryzae is the highest, including isovaleric acid (D), methyl thiocyanate, isovaleric acid (M) and 2,2,4,6,6-pentamethylheptane (D). Among the four regions, the concentration of volatile compounds in shrimp paste fermented with Mucor was the highest, including 2-methyldihydro-3(2H)-furanone (D), ethyl propionate, butyric acid (D) and 2-methyldihydro-3(2H)-furanone (M). Esters in shrimp paste are produced by the esterification of alcohols and carboxylic acids catalyzed by alcohol acyltransferases, usually showing fruity and floral aromas and able to mask the unpleasant odor caused by free fatty acids. Aldehydes in shrimp paste are produced by fatty acid oxidation and Maillard reaction during the fermentation process, which can enhance the sweetness, fruitiness, nutty flavors and caramel flavors, and contribute greatly to the main flavor of shrimp paste. Organic acids in acids, such as formic acid, propionic acid and butyric acid, are mainly produced during the fermentation process and contribute little to the aroma of shrimp paste due to their high threshold. Ketones in shrimp paste are formed by the action of enzymes in microorganisms on lipids or amino acids, and have a pleasant smell similar to cheese.

[0064] 7. Evaluation of key volatile compounds: The relative odor activity value (ROAV) of key volatile compounds in shrimp paste was calculated according to the method reported by Huang et al. (HUANG J, LIU Y, SHANG S, et al. Changes in bacterial flora and flavor of shrimp paste under different salt concentrations[J]. LWT, 2024, 205:116534). The volatile compound that contributed the most to the overall flavor of the sample was defined as ROAVstan = 100, and the ROAV values of other volatiles were calculated according to formula (3).

[0065]

[0066] Where: C i is the relative percentage of the compound to be measured; T i is the sensory threshold (μg / kg) of the compound to be measured; C stan is the relative percentage of the compound that contributes the most to the overall flavor; T stan is the sensory threshold (μg / kg) of the compound that contributes the most to the overall flavor.

[0067] To screen out the key volatile compounds in shrimp paste, the peak volume normalization method was used to quantitatively analyze the volatile compounds in fermented shrimp paste. 2-Acetyl-1-pyrroline with a low flavor threshold and high relative content was selected as the key flavor substance, and the ROAV values of other volatile compounds were calculated. Based on the substances with ROAV values ≥ 0.01 in shrimp paste, a cluster heat map of volatile compounds was drawn, and the results are as Figure 5 shown. When the ROAV value ≥ 1, it indicates that the substance is a key volatile compound in the sample, while when 0.1 < ROAV value ≤ 1, it indicates that the substance only acts as a regulator of the volatile flavor of the sample. According to Figure 5The results showed that there were 7 substances with ROAV values ​​of volatile compounds greater than 1 in the fermented shrimp paste, and their contribution to the volatile flavor of shrimp paste from high to low was 2-acetyl-1-pyrroline, n-valeraldehyde (D), 3,5,5-trimethyl-2-cyclohexene-1-one, L-carvone, methyl isobutyl ketone, ethyl 3-methylthiopropionate, and 2-propyl mercaptan. Among them, the ROAV values ​​of volatile compounds of L-carvone, ethyl 3-methylthiopropionate and 2-acetyl-1-pyrroline were the highest in the shrimp paste fermented by Aspergillus oryzae, followed by Mucor, Rhizopus oryzae and Aspergillus niger. In the shrimp paste prepared by Aspergillus oryzae fermentation, the ROAV values ​​of 3,5,5-trimethyl-2-cyclohexene-1-one, L-carvone, 2-acetyl-1-pyrroline, ethyl 3-methylthiopropionate and 2-propylmercaptan were all very high, indicating that these volatile compounds contributed the most to the volatile flavor of shrimp paste fermented by Aspergillus oryzae. Figure 5 The results also showed that methyl isobutyl ketone contributed the most to shrimp paste fermented with Mucor, n-valeraldehyde (D) contributed the most to shrimp paste fermented with Aspergillus niger, and 2-propyl mercaptan contributed less to shrimp paste fermented with Aspergillus niger and Rhizopus oryzae. Therefore, there may be differences in the microbial community and endogenous enzyme activity produced by fermenting shrimp paste with different strains, resulting in changes in the composition and content of volatile compounds in shrimp paste.

[0068] In summary, the shrimp pastes fermented by the four strains are all rich in volatile compounds with aromatic odor, and the aromatic odor of L-carvone, ethyl 3-methylthiopropionate and 2-acetyl-1-pyrroline in the shrimp paste fermented by Aspergillus oryzae is better than that of the shrimp paste fermented by Mucor, Rhizopus oryzae and Aspergillus niger.

[0069] Sensory evaluation of shrimp paste: Referring to the method reported by Duan Zerui et al. (Duan Zerui, Tian Meilan, Liu Yangliu, et al. Effect of salt addition on the physicochemical properties and bacterial diversity of shrimp paste [J]. Food Science, 2024, 45(15): 85-93), sensory evaluators who have received unified training were invited to evaluate the shrimp paste after fermentation. The specific scoring criteria are shown in Table 4.

[0070] Table 4 Sensory evaluation standard table of shrimp paste

[0071]

[0072] Data analysis: All experiments were repeated at least 3 times, and the experimental results were expressed as mean ± standard deviation. The data were plotted using Origin2021 software, and the Duncan test was used to analyze the data significance using SPSS26 software, with the significance level set at 0.05.

[0073] The results of the sensory evaluation of shrimp paste prepared by fermentation with different strains of bacteria are shown in Table 5. In the sensory evaluation of the four fermented shrimp pastes, the shrimp paste prepared by fermentation with Mucor had the highest scores in color, taste and tissue morphology. There was no significant difference in the color and taste scores of the shrimp paste prepared by fermentation with Rhizopus oryzae and Aspergillus oryzae, and both were significantly higher than the shrimp paste prepared by fermentation with Aspergillus niger. In terms of smell, the shrimp paste prepared by fermentation with Aspergillus oryzae had the highest score, while there was no significant difference in the scores of shrimp paste prepared by fermentation with Rhizopus oryzae and Mucor, but it was higher than the shrimp paste prepared by fermentation with Aspergillus niger. The results of this application show that the shrimp paste prepared by fermentation with Mucor has good color, taste and tissue state, and the content of corrupt substances is low (Table 1, Figure 2 , Table 3 Figure 1 ), but the content of volatile flavor substances with aromatic smell is low ( Figure 5 ).

[0074] Table 5 Effect of bacterial strains on the sensory quality of shrimp paste

[0075]

[0076]

[0077] In summary, the experiments revealed the effects of different strains on the physical and chemical properties of fermented shrimp paste by fermenting shrimp shells, shrimp heads and soybeans with Aspergillus oryzae, Rhizopus oryzae, Aspergillus niger and Mucor. With the extension of fermentation time, the amino nitrogen and volatile basic nitrogen contents of the four shrimp pastes gradually increased. The amino nitrogen content of shrimp paste fermented with Mucor and Rhizopus oryzae for 28 days was high, and the volatile basic nitrogen content was low. The taste of shrimp paste fermented with Mucor and Rhizopus oryzae was higher than that of other fermented shrimp pastes, and the relative odor activity values ​​of aromatic L-carvone, ethyl 3-methylthiopropionate and 2-acetyl-1-pyrroline were lower than those of shrimp paste fermented with Aspergillus oryzae. Therefore, the shrimp paste prepared by fermenting shrimp shells, shrimp heads and soybeans with Mucor has good color and umami taste.

[0078] Embodiment 3 Multi-strain koji making and fermentation of shrimp paste

[0079] Mucor, Rhizopus oryzae, and Aspergillus oryzae were selected as the strains for fermenting shrimp paste. Each strain was inoculated at a ratio of 0.125% (mass percentage) into a mixture of cooled cooked shrimp heads, shrimp shells, and soybeans, and the koji was made in a constant temperature and humidity chamber at a temperature of 28°C and a humidity of 90% for 48 hours. The orthogonal method was used to design the mixing ratio of different strains of koji, as shown in Table 6.

[0080] Table 6 Orthogonal test design Multi-strain koji making mixed fermentation shrimp paste strain koji adding ratio table (weight parts)

[0081]

[0082] According to the corresponding detection method of Example 2, the results are shown in Table 7.

[0083] Table 7 Orthogonal test results

[0084]

[0085]

[0086] Note: Ki represents the average value of the sum of the test results corresponding to the level number i in any column, and K1 value is the average value of the sum of the test results corresponding to the level 1 under each factor. For example, k1 represents the average value of the sum of the amino nitrogen content results corresponding to the Mucor proportion of 1 in the Mucor (factor) column, that is, (0.97+0.94+1.00)÷3=0.97.

[0087] As shown in Table 7, the optimal level of amino nitrogen content is No. 7, i.e., A3B1C3, indicating that the optimal process for preparing shrimp paste is Mucor koji: Rhizopus oryzae koji: Aspergillus oryzae koji = 3:1:3 (weight ratio). The larger the range, the greater the influence of the factor on the quality of shrimp paste. The order of influence of the three factors on amino nitrogen is Mucor koji (A) > Aspergillus oryzae koji (B) > Rhizopus oryzae koji (C), indicating that Mucor has the greatest influence on the amino nitrogen of shrimp paste. The optimal level of sensory evaluation is A3B1C3, indicating that the optimal process for preparing shrimp paste is Mucor koji: Rhizopus oryzae koji: Aspergillus oryzae koji = 3:1:3 (weight ratio). The order of influence of the three factors on sensory scores is Mucor koji (A) > Aspergillus oryzae koji (B) > Rhizopus oryzae koji (C), indicating that Mucor has the greatest influence on the sensory evaluation of shrimp paste.

[0088] It can also be seen from Table 7 that the shrimp paste prepared by process No. 9, i.e. A3B3C2, also performed well; followed by No. 5 (A2B3C3); No. 3 (A1B3C3); No. 1 (A1B1C1); No. 8 (A3B2C1); No. 6 (A2B3C1); No. 2 (A1B2C2); and No. 4 (A2B1C2).

[0089] According to the corresponding detection method of Example 2, the volatile compounds in the multi-strain fermented shrimp paste were determined. Figure 6 .

[0090] Depend on Figure 6 It can be seen that the ion migration time of volatile compounds in the 9 groups of shrimp paste samples is concentrated in 1.0-2.0ms, and the retention time is concentrated in 200-1800s. In addition, the concentration of most volatile compounds in shrimp paste No. 7 is higher than that in other shrimp pastes. GC-IMS was used to analyze the volatile components in shrimp paste fermented with multiple strains, and a total of 88 volatile compounds were detected. The most common types of shrimp paste are ketones (19 types), followed by aldehydes (16 types), alcohols (15 types), other substances (15 types), esters (12 types), acids (6 types) and pyrazines (5 types).

[0091] The fingerprint of volatile compounds was obtained by analyzing 88 volatile compounds detected in shrimp paste. Figure 7 As shown. The color of the spots in the figure from light to dark represents the concentration of the samples from low to high. In area A, the concentration of volatile compounds in shrimp paste No. 7 is higher than that in other shrimp pastes, including 28 volatile compounds such as methyl pyrazine, (E)-3-hexen-1-ol and methylcyclopentenolone (M). In area B, the concentration of volatile compounds in shrimp paste No. 8 is higher than that in other shrimp pastes, including 10 volatile compounds such as n-octanol, terpinolene and 2-methylpyrazine. In area C, the concentration of volatile compounds in shrimp paste No. 9 is higher than that in other shrimp pastes, including 16 volatile compounds such as (Z)-2-pentenol, 3-hydroxy-2-methyl-4-pyrone and 2,3-dimethylpyrazine.

[0092] In order to clarify the main contributing components of shrimp paste flavor, the relative odor activity method was used to determine the key volatile compounds with ROAV values ​​≥ 1 in shrimp paste (Table 8).

[0093] As shown in Table 8, a total of 10 volatile compounds with aroma were obtained in 9 samples. Among them, the ROAV values ​​of the five key volatile compounds in No. 7 shrimp paste were higher than those of other shrimp pastes, including n-pentanal monomer and n-pentanal dimer with almond aroma, n-hexanal with fat aroma, 3-pentanone (M) with ether aroma, ethyl acetate with roasted nut aroma, and 3,5,5-trimethyl-2-cyclohexene-1-one with fruit aroma. Studies have found that n-pentanal monomer and n-pentanal dimer and n-hexanal produced by the oxidation of fatty acids and Maillard reaction during the fermentation of shrimp paste contribute greatly to the overall flavor of Antarctic krill shrimp paste. 3-Pentanone produced by lipids or amino acids under the action of enzymes secreted by microorganisms and the aromatic odor of ethyl acetate produced by protein degradation and fat oxidation can improve the overall flavor of shrimp paste. Yang et al. prepared meat seasoning by optimizing the ratio of mixed fermentation of Monascus, Mucor and Bacillus subtilis. They found that mixed fermentation under the optimal combination of three strains of 1:1:1 can secrete abundant proteases.

[0094] Table 8 Key volatile compounds in shrimp paste

[0095]

[0096] To promote the formation of volatile compounds with aromatic smell such as 3,5,5-trimethyl-2-cyclohexene-1-one. The fermented shrimp paste of this embodiment is mixed and fermented under the condition of a mass ratio of Mucor koji: Rhizopus oryzae koji: Aspergillus oryzae koji of 3:1:3, which can more obviously promote the formation of key volatile compounds in the shrimp paste, and the prepared No. 7 shrimp paste has a strong aromatic smell.

[0097] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. Use of Aspergillus, Aspergillus niger, Mucor and / or Rhizopus oryzae for preparing fermented shrimp paste.

2. A fermented shrimp paste, characterized in that: The strain used for fermenting shrimp paste is at least one of Aspergillus, Aspergillus niger, Mucor and / or Rhizopus oryzae.

3. The fermented shrimp paste according to claim 2, characterized in that When the strain is a mixture of Mucor, Aspergillus oryzae and Rhizopus oryzae, the weight ratio of the three is: 3:1:3; 3:3:2; 2:3:3; 1:3:3; 1:1:1; 3:2:1; 2:3:1; 1:2:2; or 2:1:

2.

4. The fermented shrimp paste according to claim 2, characterized in that: When it is a single strain, the preparation method of fermented shrimp paste is as follows: weigh soybeans, soak them in water for more than 10 hours, drain them, steam them together with shrimp shells and shrimp heads, and then crush them with a meat grinder to obtain a mixture of soybeans, shrimp shells and shrimp heads; wherein the mass ratio of soybeans: shrimp shells: shrimp heads is 2:1:1; the mixture is inoculated with the strain, and then koji is made in a constant temperature and humidity chamber; the koji making temperature is 28°C, the humidity is 90%, and the koji making time is 48 hours; then, saline containing 10% sodium chloride is added to the obtained koji according to the mass ratio of koji: saline of 1:2, and after mixing evenly, it is transferred to a fermentation jar, fermented in a 50°C constant temperature chamber for 28 days, inactivated enzymes in a water bath at 90°C for 30 minutes, and filtered through a 10-40 mesh sieve to remove the coarse shrimp shells to obtain a shrimp paste sample; When it is a mixed strain, the preparation method of fermented shrimp paste is as follows: each strain is inoculated at a mass percentage of 0.125% into a mixture of cooled cooked shrimp heads, shrimp shells and soybeans, and koji is made in a constant temperature and humidity chamber at a temperature of 28°C and a humidity of 90% for 48 hours; then, brine containing 10% sodium chloride is added to the obtained koji at a mass ratio of 1:2 between koji and brine, and after uniform mixing, the mixture is transferred to a fermentation jar, fermented in a constant temperature chamber at 50°C for 28 days, inactivated in a water bath at 90°C for 30 minutes, and then filtered through a 10-40 mesh sieve to remove the coarse shrimp shells to obtain a shrimp paste sample.

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