Use of a bacterial strain of the species Lactobacillus fermentum in fermenting the green husk of Macadamia integrifolia

Abstract

The present invention discloses the use of a bacterial strain related to the species Lactobacillus fermentum in fermenting the green husk of Macadamia nuts, which relates to the technical field of microbial inoculants. The inventor screened and isolated a bacterial strain from the green husk of Macadamia nuts, and it was identified as belonging to the species Lactobacillus fermentum. It can improve the antioxidant activity of the green husk of Macadamia nuts through fermentation, fundamentally solve the problem of the loss of antioxidant components in the green husk of Macadamia nuts, effectively alleviate the loss of antioxidant components in plants, and has the characteristics of high efficiency, safety, and greenness. The proposed bacterial strain of the present invention can realize the "waste" utilization of the green husk of Macadamia nuts. The screened Lactobacillus fermentum can tolerate bactericidal components such as tannins.

Description

Technical Field

[0001] The invention relates to the technical field of microbial agents, and in particular to application of a bacterial strain of a fermentative Lactobacillus mucilaginosus species in fermenting macadamia nut green peel. Background Art

[0002] In recent years, the concept of "zero waste" has been gaining momentum, and the industry has been pursuing a new model of development by "squeezing out" raw materials and their by-products. Macadamia nut green peel is the green skin outside the shell, which is an agricultural by-product of the shell-nut processing process. Macadamia nut green peel contains a variety of antioxidant ingredients and can be used as a potential functional feed raw material.

[0003] Previous studies have shown that the total phenolic compounds in the water extract of macadamia nut peel are 86 mg GAE / g, the flavonoids are 30 mg RUE / g, and the proanthocyanidins are 97 mg CAE / g[1]. The half maximal inhibitory concentration (ICP) of macadamia nut peel polyphenols on DPPH free radicals and ABTS+ free radicals is 2.5477 mg / g. 50 ) were 4.13 and 112.94 mg / L, respectively, and its total antioxidant capacity was about 1.7 times that of Trolox (water-soluble vitamin E)[2]. The ethyl acetate extract had the strongest scavenging ability for DPPH free radicals and ABTS+ free radicals, and the highest reducing power. Its IC 50 The n-butanol extract had the strongest scavenging ability for O2.- free radicals, and its IC 50 The concentration of macadamia nut peel is 0.08 mg / mL, which is better than rutin at the same mass concentration [3]. Therefore, macadamia nut peel has a strong antioxidant function.

[0004] In addition, researchers have found that macadamia nut peel contains anti-inflammatory and analgesic medicinal ingredients such as tannin, arbutin, curcumin, and β-sitosterol [4], [7]. Given that macadamia nut peel has strong antioxidant properties and contains certain nutrients and medicinal ingredients, if it can be used as a functional feed for animals, the added value of the peel will be increased, achieving the goal of "turning waste into treasure."

[0005] However, macadamia nut peel is a living plant tissue. After being harvested, it undergoes processes such as baking, squeezing, and peeling. Due to physical stress, the ROS content in the tissue accumulates in large quantities, causing a large loss of antioxidant components[5]. In addition, macadamia nut peel contains high levels of bactericidal components such as tannins, making it difficult for microorganisms to survive[6].

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a bacterial strain of the species Limosilactobacillus fermentum for fermenting the green husk of Macadamia nuts, so as to solve the problems that the green husk of Macadamia nuts, as agricultural waste, has low utilization rate, high antioxidant active ingredients are easily lost, and the content of bactericidal ingredients such as tannins is high, making it difficult for microorganisms to survive.

[0008] The present invention is implemented as follows:

[0009] In a first aspect, the present invention provides the use of a bacterial strain of the species Limosilactobacillus fermentum in fermenting the green husk of Macadamia nuts.

[0010] The inventor screened and isolated a bacterial strain from the green husk of Macadamia nuts. After identification, it belongs to the species Limosilactobacillus fermentum. It can improve the antioxidant activity of the green husk of Macadamia nuts through fermentation, fundamentally solve the problem of the loss of antioxidant components in the green husk of Macadamia nuts, effectively alleviate the loss of antioxidant components in plants, and has the characteristics of high efficiency, safety, and greenness. The proposed bacterial strain of the present invention can realize the "waste" utilization of the green husk of Macadamia nuts. The screened Limosilactobacillus fermentum can tolerate bactericidal components such as tannins.

[0011] As a kind of lactic acid bacteria, Limosilactobacillus fermentum is currently widely used as a natural fermenting agent in the fields of feed and food fermentation. The inventor found that through the action of Limosilactobacillus fermentum, the antioxidant components in the green husk of Macadamia nuts can be further released and activated, the antioxidant activity of the green husk of Macadamia nuts can be improved, the problem of the loss of antioxidant components in the green husk of Macadamia nuts is fundamentally solved, the loss of antioxidant components in plants is effectively alleviated, and it has the characteristics of high efficiency, safety, and greenness.

[0012] The present invention first isolated and purified Limosilactobacillus fermentum for fermenting the green husk of Macadamia nuts from the green husk of Macadamia nuts, which helps to increase the added value of the Macadamia nut peel, promote the reduction and resource utilization of agricultural waste, and enrich the germplasm resources of antioxidant fermentation microorganisms.

[0013] In a preferred embodiment of the application of the present invention, the bacterial strain has a 16s rDNA sequence that is at least 99% identical to SEQ ID NO.1. For example, the bacterial strain has a 16s rDNA sequence that is 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, or 100% identical to SEQ ID NO.1.

[0014] In a preferred embodiment of the application of the present invention, the bacterial strain is the cell of the strain with the preservation number: CCTCC NO: M 20222085 or its progeny strain or subcloned strain.

[0015] The strain with the preservation number: CCTCC NO: M 20222085 was preserved on December 28, 2022 at the China Center for Type Culture Collection, with the preservation address: Wuhan University, Wuhan, China. The strain classification characteristics are: Limosilactobacillus fermentum ZC529. After identification, the strain of CCTCC NO: M 20222085 is a Gram-positive bacterium.

[0016] In a second aspect, the present invention provides cells of Limosilactobacillus fermentum or its progeny strains or subcloned strains preserved under the preservation number CCTCC NO: M20222085.

[0017] In a third aspect, the present invention also provides the use of a bacterial strain of Limosilactobacillus fermentum in the preparation of a fermenting agent for Macadamia integrifolia pericarp.

[0018] It should be noted that the above-mentioned fermenting agent includes, but is not limited to, liquid preparations, solid fermenting agents, paste fermenting agents, suspensions, emulsions, sprays or semi-solid fermenting agents.

[0019] In a preferred embodiment of the application of the present invention, the Limosilactobacillus fermentum is the Limosilactobacillus fermentum preserved under the preservation number CCTCC NO: M20222085 or its progeny strains.

[0020] In a fourth aspect, the present invention also provides a fermenting agent, which includes: cells of Limosilactobacillus fermentum or its progeny strains or subcloned strains preserved under the preservation number CCTCC NO: M 20222085.

[0021] It should be noted that the above-mentioned fermenting agent includes, but is not limited to, liquid preparations, solid fermenting agents, paste fermenting agents, suspensions, emulsions, sprays or semi-solid fermenting agents.

[0022] The above-mentioned fermenting agent can be used for feeding livestock, poultry or aquatic animals, improving the antioxidant capacity of livestock, poultry or aquatic animals, adjusting the intestinal flora, and stopping diarrhea and sterilizing. Aquatic animals are, for example, selected from aquatic animals such as shrimp (especially krill), fish, crabs, shellfish, etc.

[0023] In a fifth aspect, the present invention also provides a composition, which includes: the above-mentioned fermenting agent and Macadamia integrifolia pericarp. The Macadamia integrifolia pericarp includes, but is not limited to, fresh or slightly deteriorated Macadamia integrifolia pericarp, or freeze-dried and powdered Macadamia integrifolia pericarp powder.

[0024] The composition includes, but is not limited to, a blend of the fermenting agent and Macadamia integrifolia pericarp, a fermentation product after fermentation for a period of time, etc.

[0025] Sixth aspect, the present invention also provides a fermented product of macadamia nut green peel, which is obtained by fermenting macadamia nut green peel with Lactobacillus mucilaginosus fermentum deposited under the accession number CCTCC NO: M 20222085 or its progeny strains.

[0026] Compared with before fermentation, the total antioxidant capacity in this fermented product is significantly improved. After fermentation, the tannin content of macadamia nut green peel decreases significantly, while the flavonoid content increases. This helps to improve the palatability of the fermented product of macadamia nut green peel (reduce the bitter taste).

[0027] In an optional embodiment, the fermentation conditions are 28 - 32 °C and the fermentation time is 3 - 10 d.

[0028] Seventh aspect, the present invention also provides a feed additive, which comprises the above-mentioned fermented product of macadamia nut green peel or the above-mentioned microbial agent.

[0029] When the microbial agent is directly used as a feed additive, it can be used to feed livestock, poultry or aquatic animals, and can also be used to prepare probiotics.

[0030] When the fermented product of macadamia nut green peel is used as a feed additive, it can be used to prepare roughage for monogastric and ruminant animals.

[0031] The present invention has the following beneficial effects:

[0032] The present invention screens and isolates a bacterial strain from macadamia nut green peel. After identification, it belongs to the species Lactobacillus mucilaginosus fermentum. It can improve the antioxidant activity of macadamia nut green peel through fermentation, fundamentally solve the problem of the loss of antioxidant components in macadamia nut green peel, effectively alleviate the loss of antioxidant components in plants, and has the characteristics of high efficiency, safety and greenness. The proposal of the bacterial strain of the present invention can realize the "waste" utilization of macadamia nut green peel. The screened Lactobacillus mucilaginosus fermentum can tolerate bactericidal components such as tannins.

[0033] The inventor found that fermenting macadamia nut green peel with Lactobacillus mucilaginosus fermentum can further release and activate the antioxidant components in macadamia nut green peel, improve the antioxidant activity of macadamia nut green peel, fundamentally solve the problem of the loss of antioxidant components in macadamia nut green peel, effectively alleviate the loss of antioxidant components in plants, and has the characteristics of high efficiency, safety and greenness.

[0034] The present invention first isolates and purifies Lactobacillus mucilaginosus fermentum for fermenting macadamia nut green peel from macadamia nut green peel, which helps to increase the added value of macadamia nut peel, promote the reduction and resource utilization of agricultural waste, and enrich the germplasm resources of antioxidant fermenting microorganisms. Description of the Drawings

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0036] Figure 1 It is a flow chart of the screening and preparation method of Lactobacillus mucosae fermentum.

[0037] Figure 2 It is the effect of Lactobacillus mucosae fermentum on the total antioxidant capacity of MRS and the green peel of Macadamia nuts; (Note: "*" indicates significant difference compared with the control, P < 0.05);

[0038] Figure 3 It is the morphological, physiological and biochemical identification results of Lactobacillus mucosae fermentum. Detailed implementation manners

[0039] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0040] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0041] Embodiment 1

[0042] The flow chart of the screening and isolation of strains is referred to Figure 1 as shown.

[0043] (1) Materials, culture media and fermentation raw materials involved in the experiment:

[0044] The bacterial strain enrichment solution is sterile pure water (pH 7.0 ± 0.5).

[0045] Culture medium (g / L): peptone 10g, beef extract powder 8g, yeast powder 4g, glucose 20g, dipotassium hydrogen phosphate 2g, ammonium citrate hydrogen 2g, sodium acetate 5g, magnesium sulfate 0.2g, manganese sulfate 0.04g, Tween - 80 1ml; pH value 5.7 ± 0.2; autoclaved at 115°C for 15 min; 18g of agar powder is added to the solid culture medium correspondingly.

[0046] The fermentation raw material is: fresh green peel of Macadamia nuts, crushed to less than 1 cm 2 fragments.

[0047] (2) Collect fresh or slightly deteriorated Macadamia integrifolia pericarp as samples. According to the ratio of sample∶sterile water = 1 g∶10 mL, immerse the samples in the bacterial enrichment solution, shake well, and conduct enrichment culture at room temperature for 7 days.

[0048] (3) Absorb the sample solution from the bottom of the enrichment culture solution. Dilute the sampled solution and spread it on the MRS solid plate, and culture it anaerobically at 37°C for 1 - 7 days. After the colonies grow, pick the strains with different colony morphologies respectively for isolation and purification, and inoculate the purified strains into the MRS liquid medium, and culture them statically at 37°C for 24 h to prepare the seed solution. This step is used to culture the epiphytic lactic acid bacteria on the Macadamia integrifolia pericarp and isolate and purify the cultured lactic acid bacteria.

[0049] (4) Take the seed solution and inoculate it into the MRS liquid culture at an inoculation amount of 2%, and culture it statically at 37°C for 24 h to prepare the fermentation broth. After shaking and mixing the fermentation broth, centrifuge it at 8000×g at 4°C for 5 min, take the supernatant, and detect the total antioxidant capacity (T-AOC) of the fermentation supernatant by the FRAP method, using the blank uninoculated MRS medium as the control. This step is used to screen out the lactic acid bacteria with antioxidant properties.

[0050] (5) Select the strains with increased T-AOC, inoculate them into the MRS medium, culture them until the stationary phase, adjust the bacterial liquid concentration to ×10 8 CFU / mL, inoculate them into the fresh Macadamia integrifolia pericarp to be fermented, with an inoculation amount of 4 mL / 100 g, add 0.9% cellulase (enzyme activity ≥ 2×10 4 U / g), adjust the water content to 60%, mix well, pack them in a sterile fermentation bag, evacuate the air, and the fermentation temperature is 30°C; this step is used to screen out the fermentation strains that can survive normally and have high antioxidant properties in the Macadamia integrifolia pericarp.

[0051] (6) Take the sample at 0 d of fermentation, store it at -20°C, and regard it as the original control group. Take samples after continuous fermentation for 7 d, detect the T-AOC of the samples, and compare with the control group.

[0052] Repeat the same operation until the fermentation strain with the strongest antioxidant property is screened out.

[0053] (7) Select the fermentation strain with the strongest antioxidant property for species identification by 16S rDNA sequencing and preservation.

[0054] Analyze and compare the changes in the T-AOC values of the MRS medium before and after inoculating the case strain in step (5), and analyze and compare the changes in the T-AOC values of the Macadamia integrifolia pericarp at the initial stage of inoculating the case strain and after 7 d of fermentation in steps (5) and (6).

[0055] Figure 2It can be seen that the inventor screened out 1 antioxidant strain that can ferment the green skin of Macadamia nuts, which can increase the total antioxidant capacity of MRS medium by 10.51% and increase the total antioxidant capacity of fermented green skin of Macadamia nuts by 158.35%.

[0056] Example 2

[0057] In this example, the strain isolated in Example 1 was subjected to Gram staining and physiological and biochemical identification. The identification results refer to Figure 3 as shown Figure 3 in A: colony morphology; B: Gram staining; C: physiological and biochemical reactions.

[0058] The screened bacterium is a Gram-positive bacterium, positive for maltose, sucrose, 1% hippurate, raffinose, inulin, catalase and glucose gas production. After 16S rDNA sequencing and comparison, the screened strain is Lactobacillus mucosae fermentum. The 16S rDNA is as shown in SEQ ID NO: 1.

[0059] Experimental Example 1

[0060] In this experimental example, the tannins before and after fermentation were detected.

[0061] The strain with the preservation number: CCTCC NO: M 20222085 screened in Example 1 was adjusted to a bacterial liquid concentration of ×10 8 CFU / mL, inoculated on the green skin of Macadamia nuts, the inoculation amount was 4 mL / 100 g, the water content was 60%, mixed evenly, packed in a sterile fermentation bag and evacuated, the fermentation temperature was 30 °C, and the fermentation was carried out for 7 days. According to the method of GB / T 27985-2011[8], the tannin content in the fermented green skin of Macadamia nuts was determined, and the original unfermented green skin of Macadamia nuts was used as a control.

[0062] The results showed that the tannin content in the original green skin of Macadamia nuts was 1.85%. After fermentation treatment, the tannin content decreased to 0.76%. Fermentation treatment with the strain provided in Example 1 of the present invention can reduce the tannin content in the green skin of Macadamia nuts and improve the palatability of the green skin of Macadamia nuts.

[0063] Experimental Example 2

[0064] In this experimental example, the flavonoids before and after fermentation were detected.

[0065] The strain with the preservation number: CCTCC NO: M 20222085 screened in Example 1 was adjusted to a bacterial liquid concentration of ×10 8 CFU / mL, inoculated on the green skin of Macadamia nuts, the inoculation amount was 4 mL / 100 g, and 0.9% cellulase (enzyme activity ≥ 2×10 4Adjust the water content to 60% (U / g), mix evenly, pack it in a sterile fermentation bag, evacuate it, ferment at 30°C for 7 days. According to the report by Yan Zankai [9], the total flavonoid content of the fermented macadamia nut green peel was determined by colorimetric assay, with the original unfermented macadamia nut green peel as the control.

[0066] The results showed that the total flavonoid content in the original macadamia nut green peel was 1.83 mg / g. After fermentation, the total flavonoid content reached 3.80 mg / g. Fermentation with the strain provided in Example 1 of the present invention can increase the total flavonoid content in the macadamia nut green peel, improving its nutritional value and antioxidant properties.

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[0068] 2: Zhang Ming, Shuai Xixiang, Du Liqing, Tu Xinghao. Optimization of the Extraction Process of Polyphenols from Macadamia Nut Green Peel and Its Antioxidant Activity [J]. Science and Technology of Food Industry, 2017, 38(22):195 - 199.

[0069] 3: Guo Gangjun, Hu Xiaojing, Fu Jiarong, Ma Shangxuan, Xu Rong, Huang Kechang, Peng Zhidong, He Xiyong, Zou Jianyun. Analysis of the Functional Components and Antioxidant Activity and Their Correlation of Stepwise Extracts of Macadamia Nut Green Peel with Different Polar Solvents [J]. Food Science, 2021, 42(07):74 - 82.

[0070] 4: Zhang Mingkai, Yang Weihai, Zeng Hui, et al. Analysis of the Main Functional Components in Macadamia Nut Pericarp [J]. Chinese Journal of Tropical Agriculture, 2011, 31(05):73 - 75.

[0071] 5: Ban Zhaojun, Guan Junfeng, Li Li, etc. Research Overview on the Generation of Reactive Oxygen Species and Antioxidant Mechanisms in Plants under Abiotic Stress [J]. China Fruit & Vegetable, 2012, 169(05):40 - 47.

[0072] 6: Qiu Jianqiang, Huang Wen, Wang Yuxi, etc. Research Progress on the Regulation of Animal Intestinal Microflora by Tannins [J]. Chinese Journal of Animal Nutrition, 2023:1 - 10.

[0073] 7: Shi Rui, Xiong Zhi, Ke Jin, Liu Shaoxiong, Shi Zhuogong, Kan Huan. Preparation method of macadamia husk extract and lotion [P]. Yunnan: CN102727412A, 2012-10-17.

[0074] 8: General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Standardization Administration of China. Determination of tannins in feeds - Spectrophotometric method: GB / T27985-2011 [S]. Beijing: Standards Press of China.

[0075] 9: Yan Zankai. Method for determining flavonoid content in plants by ultraviolet spectrophotometry [J]. Food Research and Development, 2007, No.142(09): 164-167.

[0076] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of Lactobacillus mucosae fermentum ( Limosilactobacillus fermentum ) in fermenting the green husk of Macadamia integrifolia Characterized in that, the Limosilactobacillus fermentum is the strain with the preservation number: CCTCC NO: M 20222085.

2. A Limosilactobacillus fermentum preserved with the preservation number CCTCC NO: M 20222085.

3. Use of a bacterial strain of Limosilactobacillus fermentum in the preparation of a fermenting agent for Macadamia integrifolia pericarp, Characterized in that, the Limosilactobacillus fermentum is the Limosilactobacillus fermentum preserved with the preservation number CCTCC NO: M 20222085.

4. A fermenting agent, Characterized in that, it comprises: Limosilactobacillus fermentum preserved with the preservation number CCTCC NO: M 20222085.

5. A composition, Characterized in that, it comprises: the fermenting agent according to claim 4 and Macadamia integrifolia pericarp.

6. A fermentation product of Macadamia integrifolia pericarp, Characterized in that, it is obtained by fermenting Macadamia integrifolia pericarp with Limosilactobacillus fermentum preserved with the preservation number CCTCC NO: M 20222085.

7. The fermentation product of Macadamia integrifolia pericarp according to claim 6, Characterized in that, the fermentation conditions are 28 - 32 °C and the fermentation time is 3 - 10 d.

8. A feed additive, Characterized in that, it comprises the fermentation product of Macadamia integrifolia pericarp according to any one of claims 6 - 7 or the fermenting agent according to claim 4.

9. The feed additive according to claim 8, Characterized in that, the feed additive is a poultry and livestock feed additive or an aquatic animal feed additive.

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