Method for lysing marine purple photosynthetic bacteria

Abstract

The present invention relates to a method for lysing marine purple photosynthetic bacteria, said method comprising a step for obtaining a lysate of marine purple photosynthetic bacteria by lysing the marine purple photosynthetic bacteria in an acetone solvent, wherein the lysis suppresses a change in amino acid composition or carotenoid composition in the marine purple photosynthetic bacteria.

Description

Method for disrupting marine purple photosynthetic bacteria

[0001] This invention relates to a method for disrupting marine purple photosynthetic bacteria.

[0002] Marine purple photosynthetic bacteria are bacteria that can utilize seawater, nitrogen, carbon dioxide, and light, which are abundant on Earth, for their growth. It is expected that marine purple photosynthetic bacteria can contribute to the preservation of the global environment by enabling the sustainable production of nitrogen and carbon-containing substances using natural resources. For example, Patent Document 1 describes that marine purple photosynthetic bacteria can be used to produce aquaculture feed with a high protein content. Furthermore, Patent Document 2 describes that marine purple photosynthetic bacteria can be used to produce agricultural fertilizers with a high nitrogen content, and that marine purple photosynthetic bacteria can be used to produce aquaculture feed with a high protein content.

[0003] International Publication No. 2023 / 214591 International Publication No. 2023 / 214592

[0004] In order to efficiently utilize the amino acids and / or carotenoids produced by marine purple photosynthetic bacteria, there is a need for a crushing method that can suppress changes in the composition of amino acids and / or carotenoids in marine purple photosynthetic bacteria when the bacteria are crushed. One of the problems that the present invention aims to solve is to provide a crushing method that suppresses changes in the composition of amino acids in marine purple photosynthetic bacteria when the bacteria are crushed. Another problem that the present invention aims to solve is to provide a crushing method that suppresses changes in the composition of carotenoids in marine purple photosynthetic bacteria when the bacteria are crushed. Another problem that the present invention aims to solve is to provide a crushing method that suppresses changes in the composition of amino acids and carotenoids in marine purple photosynthetic bacteria when the bacteria are crushed.

[0005] As a result of diligent research by the inventors, they discovered that disrupting marine purple photosynthetic bacteria in acetone solvent suppresses changes in the amino acid composition or carotenoid composition within the marine purple photosynthetic bacteria, thus completing the present invention.

[0006] In other words, the present invention includes the following embodiments: [1] A method for crushing marine purple photosynthetic bacteria, comprising the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria, wherein the crushing suppresses changes in the composition of amino acids or carotenoids in the marine purple photosynthetic bacteria. [2] The method for crushing marine purple photosynthetic bacteria according to [1], wherein the crushing of marine purple photosynthetic bacteria is carried out by immersing marine purple photosynthetic bacteria in an acetone solvent. [3] The method for crushing according to [1] or [2], wherein the change in the composition of amino acids is a change in the ratio of the content of each natural amino acid contained in the crushed product compared with the ratio of the content of each natural amino acid contained in the marine purple photosynthetic bacteria. [4] The method for crushing according to any one of [1] to [3], wherein the change in the composition of amino acids is a change in the total amount of amino acids and / or the total amount of free amino acids contained in the marine purple photosynthetic bacteria. [5] The crushing method according to any one of [1] to [4], wherein the amount of amino acids contained in the crushed material is 85% by mass or more of the total amount of amino acids contained in marine purple photosynthetic bacteria. [6] The crushing method according to any one of [1] to [5], wherein the amount of free amino acids contained in the crushed material is 85% by mass or more of the total amount of free amino acids contained in marine purple photosynthetic bacteria. [7] The crushing method according to any one of [1] to [6], wherein the change in the composition of the carotenoids is a change in the total amount of carotenoids contained in marine purple photosynthetic bacteria. [8] The crushing method according to any one of [1] to [7], wherein the total amount of carotenoids contained in the crushed material is 90% by mass or more of the total amount of carotenoids contained in marine purple photosynthetic bacteria. [9] The crushing method according to any one of [1] to [8], wherein the carotenoid is one or more selected from the group consisting of spheroidenone, spheroidene, dimethylspheroidenone, 3,4-dihydroxyspheroidenone, and neurospolene.

[10] The crushing method according to any one of [1] to [9], further comprising the step of drying the crushed material.

[11] The crushing method according to any one of [1] to

[10] , wherein the crushed material is for use as a nutrient.

[12] The crushing method according to any one of [1] to

[11] , wherein the nutrient is for use as a fertilizer, feed, food, or culture medium.

[13] A method for suppressing changes in the amino acid composition or carotenoid composition of marine purple photosynthetic bacteria, comprising the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria.

[0007] The present invention also includes the following embodiments: [1A] A method for crushing marine purple photosynthetic bacteria, comprising the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria, wherein the crushing suppresses a change in the amino acid composition in the marine purple photosynthetic bacteria. [2A] The method for crushing marine purple photosynthetic bacteria according to [1A], wherein the crushing of marine purple photosynthetic bacteria is carried out by immersing marine purple photosynthetic bacteria in an acetone solvent. [3A] The method for crushing according to [1A] or [2A], wherein the change in amino acid composition is a change in the ratio of the content of each natural amino acid contained in the crushed product compared with the ratio of the content of each natural amino acid contained in the marine purple photosynthetic bacteria. [4A] The method for crushing according to any one of [1A] to [3A], wherein the change in amino acid composition is a change in the total amount of amino acids and / or the total amount of free amino acids contained in the marine purple photosynthetic bacteria. [5A] The crushing method according to any one of [1A] to [4A], wherein the amount of amino acids contained in the crushed material is 85% by mass or more of the total amount of amino acids contained in marine purple photosynthetic bacteria. [6A] The crushing method according to any one of [1A] to [5A], wherein the amount of free amino acids contained in the crushed material is 85% by mass or more of the total amount of free amino acids contained in marine purple photosynthetic bacteria. [7A] The crushing method according to any one of [1A] to [6A], further comprising the step of drying the crushed material. [8A] The crushing method according to any one of [1A] to [7A], wherein the crushed material is for use as a nutrient. [9A] The crushing method according to [8A], wherein the nutrient is for use as fertilizer, feed, food, or culture medium.

[0008] According to the present invention, it is possible to provide a method for crushing marine purple photosynthetic bacteria in which changes in the amino acid composition or carotenoid composition within the marine purple photosynthetic bacteria are suppressed when the bacteria are crushed.

[0009] The results of amino acid analysis for AF1-AF4 in the examples are shown. The results of amino acid analysis for AF5-AF7 in the examples are shown. The results of culturing the lysate obtained by high-pressure lysation of marine purple photosynthetic bacteria are shown. The results of culturing the lysate obtained by ultrasonic lysation of marine purple photosynthetic bacteria are shown. The results of culturing the lysate obtained by acetone lysation of marine purple photosynthetic bacteria are shown. The results of comparing the total inorganic nitrogen content of marine purple photosynthetic bacteria are shown. The results of amino acid analysis for different lysation methods of marine purple photosynthetic bacteria are shown. The results of carotenoid peak measurement are shown. The results of quantitative analysis of pigment released during acetone lysation are shown. The color changes of acetone cell suspension (upper panel) and extract (lower panel) are shown. The results of measuring the relative absorbance of the supernatant after 2.5 hours following resuspension in acetone are shown. The absorption spectrum of the supernatant after acetone lysation is shown.

[0010] The embodiments of the present invention will be described below, but the present invention is not limited by the following examples.

[0011] This disclosure relates to a method for pulverizing marine purple photosynthetic bacteria, comprising the step of pulverizing marine purple photosynthetic bacteria in an acetone solvent to obtain a pulverized product of marine purple photosynthetic bacteria, wherein the pulverization suppresses changes in the amino acid composition of the marine purple photosynthetic bacteria. Furthermore, this disclosure relates to a method for pulverizing marine purple photosynthetic bacteria, comprising the step of pulverizing marine purple photosynthetic bacteria in an acetone solvent to obtain a pulverized product of marine purple photosynthetic bacteria, wherein the pulverization suppresses changes in the carotenoid composition of the marine purple photosynthetic bacteria. Furthermore, this disclosure relates to a method for pulverizing marine purple photosynthetic bacteria, comprising the step of pulverizing marine purple photosynthetic bacteria in an acetone solvent to obtain a pulverized product of marine purple photosynthetic bacteria, wherein the pulverization suppresses changes in the amino acid and carotenoid composition of the marine purple photosynthetic bacteria. Marine purple photosynthetic bacteria will be described below.

[0012] (Marine Purple Photosynthetic Bacteria) Purple photosynthetic bacteria can be broadly classified into freshwater purple photosynthetic bacteria and marine purple photosynthetic bacteria depending on their habitat, but in this embodiment, marine purple photosynthetic bacteria that inhabit seawater areas are used. Marine purple photosynthetic bacteria can perform non-oxygen-evolving photosynthesis using carbon dioxide under near-infrared light and fix atmospheric nitrogen with nitrogenase. Marine purple photosynthetic bacteria can be cultured using natural seawater, which is cheaper to obtain than freshwater. Examples of marine purple photosynthetic bacteria include marine purple sulfur bacteria and marine purple non-sulfur bacteria. Purple sulfur bacteria are bacteria that perform photosynthesis using near-infrared light and grow photoautotrophically in the presence of hydrogen, sulfides, and carbon dioxide. Purple non-sulfur bacteria are photosynthetic bacteria that grow photoheterotrophically in the presence of organic matter, etc.

[0013] As for marine purple sulfur bacteria, there are bacteria of the genus Allochromium (Allochromium Examples of bacteria include those of the genus Ectothiodospira, Halochromium, Halochromium, Marichromium, Thiocapsa, Thiohalocapsa, and Thiophaeococcus. Examples of marine purple nonsulfur bacteria include those of the genus Rhodobaca, Rhodobacter, Rhodobium, Afifella (Rhodobium), Rhodothalassium, Rhodovulum, and Roseospira.

[0014] Furthermore, examples of marine purple photosynthetic bacteria include those disclosed in PLOS ONE | DOI: 10.1371 / journal.pone. 0160981. Specifically, these include the following purple photosynthetic bacteria that inhabit marine environments, as disclosed in Table 1 of that paper. Marine purple photosynthetic bacteria may be any marine purple photosynthetic bacteria listed as Organism in Table 1 below, specifically: Thiohalocapsa marina, Thiophaeococcus mangrovi, Marichromium beheemlicum, Afifella marina, Rhodovulum euryhalinum, Rhodovulum imhoffii, Rhodovulum sulfidophyllum, Rhodovulum tesquicola, Rhodovulum visakhapatnamense, Roseospira It may be marina and Roseospira goensis, and if the classification changes and the bacterial name changes, it may be marine purple photosynthetic bacteria as the new name for the bacteria. It may also be bacteria of the genera Thiohalocapsa, Thiophaeococcus, Marichromium, Afifella, Rhodovulum, Roseospira and Roseospira.

[0015]

[0016] Examples of marine purple sulfur bacteria include *Marichromium beeemlicum*, *Thiohalocapsa marina*, and *Thiophaeococcus mangrovi*, while examples of marine purple non-sulfur bacteria include *Afifella pfennigii* (Rhodobium pfennigii), *Afifella marina* (Rhodobium marinum), *Rhodovulum euryhalinum*, *Rhodovulum imhoffii*, *Rhodovulum sulfidophyllum*, *Rhodovulum tesquicola*, and *Rhodovulum*. Examples include visakhpatnamense, Roseospira goensis, and Roseospira marina. The marine purple photosynthetic bacteria used in this embodiment may be marine purple photosynthetic bacteria isolated from the sea off Kyoto among the above species, for example, bacteria of the genus Marichromatium. These marine purple photosynthetic bacteria may be obtained from each depositary institution through prescribed procedures. The marine purple photosynthetic bacteria described above may also be mutant strains thereof. Mutant strains include those obtained by genetic methods, such as recombination, transduction, and / or transformation.

[0017] In this embodiment, it is preferable to use marine purple photosynthetic bacteria that can grow under photosynthetic heterotrophic or photoautotrophic conditions, and it is more preferable to use Rhodovulum sulfidophyllum (R. sulfidophyllum).

[0018] (Method for disrupting marine purple photosynthetic bacteria) The method for disrupting marine purple photosynthetic bacteria according to this embodiment includes the step of disrupting marine purple photosynthetic bacteria in an acetone solvent to obtain a product of disrupted marine purple photosynthetic bacteria. The step of disrupting marine purple photosynthetic bacteria in an acetone solvent to obtain a product of disrupted marine purple photosynthetic bacteria will be described below. The product of disrupted marine purple photosynthetic bacteria obtained by disrupting marine purple photosynthetic bacteria in an acetone solvent will also be simply referred to as "disrupted product" below.

[0019] (Step to obtain crushed marine purple photosynthetic bacteria) For crushing marine purple photosynthetic bacteria, marine purple photosynthetic bacteria that have been cultured under artificial light suitable for photosynthesis and then collected may be used. That is, the method for crushing marine purple photosynthetic bacteria of this embodiment may further include the steps of culturing marine purple photosynthetic bacteria and collecting marine purple photosynthetic bacteria, which will be described later.

[0020] The crushed material is obtained by crushing marine purple photosynthetic bacteria in an acetone solvent. The acetone solvent used for crushing may be acetone alone, or a mixture of acetone and other solvents. Examples of solvents other than acetone include protic polar solvents and aprotic polar solvents. Examples of protic polar solvents include alcoholic solvents and water. When a mixture of acetone and other solvents is used for crushing, the volume ratio of acetone to other solvents (acetone / other solvent) may be 0.1 or more, 0.1 to 10, 0.5 to 5, or 1 to 2.5. In addition, one or more types of solvents other than acetone may be used. From the viewpoint of suppressing changes in the amino acid composition contained in marine purple photosynthetic bacteria, the acetone solvent used for crushing is preferably acetone alone, or a mixture of acetone and water, and more preferably acetone alone.

[0021] The disruption of marine purple photosynthetic bacteria can be carried out by bringing the marine purple photosynthetic bacteria into contact with an acetone solvent, and is not particularly limited, but may be carried out by immersing the marine purple photosynthetic bacteria in an acetone solvent, or by immersing the marine purple photosynthetic bacteria in an acetone solvent while stirring.

[0022] The amount of acetone solvent used to disrupt marine purple photosynthetic bacteria is not particularly limited, but it is preferably 0.5 mL or more, more preferably 1.0 mL or more, and even more preferably 1.5 mL or more per gram of marine purple photosynthetic bacteria. There is no particular upper limit to the amount of acetone solvent. Furthermore, the mass of marine purple photosynthetic bacteria is preferably the mass based on cell fresh weight (CFW, viable cell weight).

[0023] The temperature at which the marine purple photosynthetic bacteria are crushed is not particularly limited, but is preferably 10°C to 55°C, more preferably 15°C to 40°C, and even more preferably 20°C to 30°C. The time for immersing the marine purple photosynthetic bacteria in acetone solvent is not particularly limited, but may be 10 seconds or more, 20 seconds or more, 30 seconds or more, 1 minute or more, 5 minutes or more, 10 minutes or more, 20 minutes or more, or 30 minutes or more, and may be 3 hours or less, 2 hours or less, or 1 hour or less. Furthermore, the time for immersing the marine purple photosynthetic bacteria in acetone solvent is not particularly limited, but is preferably 10 seconds to 3 hours, more preferably 20 seconds to 2 hours, even more preferably 30 seconds to 1 hour, and even more preferably 30 minutes to 1 hour.

[0024] When crushing marine purple photosynthetic bacteria by stirring in acetone solvent, the stirring speed of the marine purple photosynthetic bacteria in the acetone solvent is not particularly limited, but is preferably 10 rpm to 1000 rpm, more preferably 30 rpm to 750 rpm, even more preferably 50 rpm to 650 rpm, and even more preferably 50 rpm to 250 rpm. The stirring time of the marine purple photosynthetic bacteria in the acetone solvent is preferably 10 seconds to 3 hours, preferably 20 seconds to 2 hours, and even more preferably 30 seconds to 1 hour.

[0025] The method for disrupting marine purple photosynthetic bacteria in this embodiment can completely disrupt the marine purple photosynthetic bacteria even when the disruption time in acetone solvent is short (for example, a few seconds), and is a method with higher disruption efficiency compared to conventional disruption methods (for example, high-pressure disruption and ultrasonic disruption).

[0026] The fact that marine purple photosynthetic bacteria have been crushed and become a fragment can be confirmed, for example, by observing that marine purple photosynthetic bacteria do not grow when the fragment is cultured using the method described in the examples. Furthermore, the fragment only needs to contain components derived from marine purple photosynthetic bacteria, but may also contain other components. Examples of other components include components derived from the culture medium used in the process of culturing marine purple photosynthetic bacteria, and components derived from the acetone solvent used in the process of obtaining the fragment of marine purple photosynthetic bacteria.

[0027] The method for disrupting marine purple photosynthetic bacteria according to this embodiment can suppress changes in the amino acid composition within the marine purple photosynthetic bacteria. Since the amino acids contained in the marine purple photosynthetic bacteria constitute the amino acids contained in the disrupted material, the amino acid composition remains unchanged before and after disruption of the marine purple photosynthetic bacteria, thus suppressing changes in the amino acid composition within the marine purple photosynthetic bacteria. Alternatively, the amount of amino acids contained in the marine purple photosynthetic bacteria may directly correspond to the amount of amino acids contained in the disrupted material. The indicator of the change in the amino acid composition within the marine purple photosynthetic bacteria may be the proportion and / or amount of each amino acid. In this embodiment, "changes in the amino acid composition in marine purple photosynthetic bacteria" may refer to, for example, (1) changes in the ratio of the content of each natural amino acid contained in the crushed material compared to the ratio of the content of each natural amino acid contained in marine purple photosynthetic bacteria, (2) changes in the content of natural amino acids contained in the crushed material compared to the content of natural amino acids contained in marine purple photosynthetic bacteria, or (3) changes in the total amount of amino acids and / or total free amino acids contained in the crushed material compared to the total amount of amino acids and / or total free amino acids contained in marine purple photosynthetic bacteria. Furthermore, suppressing changes in the amino acid composition in marine purple photosynthetic bacteria may mean that changes in the amino acid composition contained in the crushed material are suppressed compared to the amino acid composition contained in marine purple photosynthetic bacteria. Furthermore, suppressing changes in the amino acid composition of marine purple photosynthetic bacteria may mean that, when comparing the amino acid composition contained in the marine purple photosynthetic bacteria before crushing by the crushing method of this embodiment with the amino acid composition contained in the crushed material, the amino acid composition has not changed, or the change is small. The amino acid composition of the marine purple photosynthetic bacteria before crushing by the crushing method of this embodiment may be determined by analyzing the amino acid composition of freeze-dried marine purple photosynthetic bacteria. Freeze-drying can supply a sample from which the amino acids in the marine purple photosynthetic bacteria can be measured without affecting the amino acid composition contained in the marine purple photosynthetic bacteria.In this embodiment, "change in amino acid composition in marine purple photosynthetic bacteria" may refer to the change when comparing the amino acid composition contained in the marine purple photosynthetic bacteria before pulverization by the pulverization method of this embodiment with the amino acid composition contained in the pulverized material. The same applies to the following descriptions of amino acid composition and carotenoid composition. As demonstrated in the examples described later, the pulverization method for marine purple photosynthetic bacteria of this embodiment can suppress changes in amino acid composition in the sense described above, compared to conventional methods of bacterial pulverization such as high-pressure pulverization and ultrasonic pulverization.

[0028] The amino acid composition contained in marine purple photosynthetic bacteria before lysation by the marine purple photosynthetic bacteria lysation method, or in the lysated material, can be analyzed, for example, by post-columnarin hydrin derivatization of the amino acids in the marine purple photosynthetic bacteria before lysation by the marine purple photosynthetic bacteria lysation method, or in the lysated material. More specifically, for example, as described in the examples, the amino acid composition can be measured by freeze-drying the marine purple photosynthetic bacteria before lysation by the marine purple photosynthetic bacteria lysation method, preparing the lysated material, and then post-columnarin hydrin derivatization of the amino acids contained in the prepared material. Here, for the viewpoint of measurement efficiency, the amino acid composition contained in the lysated material may be measured using dried lysated material obtained by a drying process of the lysated material described later. The amino acid composition in marine purple photosynthetic bacteria before lysation by the marine purple photosynthetic bacteria lysation method may be data measured each time, an average of multiple measurements, or a predetermined measured value.

[0029] In this embodiment, with regard to the suppression of changes in the amino acid composition in marine purple photosynthetic bacteria, it may also mean that the change in the amino acid composition in marine purple photosynthetic bacteria is suppressed when compared with the amino acid composition in the crushed material after crushing. In other words, it will be explained that the change in the ratio of the content of each natural amino acid contained in the crushed material is suppressed compared with the ratio of the content of each natural amino acid contained in marine purple photosynthetic bacteria. The ratio of the content of each natural amino acid can also be rephrased as the ratio of the content between natural amino acids. In this specification, natural amino acids mean glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), histidine (His), glutamic acid (Glu), aspartic acid (Asp), glutamine (Gln), asparagine (Asn), cysteine ​​(Cys), methionine (Met), lysine (Lys), arginine (Arg), and proline (Pro). Natural amino acids are usually L-type amino acids. Suppression of changes in the ratio of each natural amino acid content in the crushed material may mean that, when comparing the ratio of each natural amino acid content to the total amino acids contained in marine purple photosynthetic bacteria with the ratio of each natural amino acid content to the total amino acids contained in the crushed material, the ratio of each natural amino acid content to the total amino acids has not changed, or has changed only slightly. The ratio of a specific natural amino acid content to the total amino acids contained in marine purple photosynthetic bacteria refers to the ratio when comparing the content of two or more natural amino acids contained in marine purple photosynthetic bacteria. The ratio of each natural amino acid content to the total amino acids contained in the crushed material refers to the ratio when comparing the content of two or more natural amino acids contained in the crushed material. The change in the ratio of each natural amino acid content may be calculated as (ratio of each natural amino acid content to the total amino acids contained in the crushed material) / (ratio of each natural amino acid content to the total amino acids contained in marine purple photosynthetic bacteria).

[0030] This section explains how the change in the content of natural amino acids in the crushed material is suppressed compared to the content of natural amino acids in marine purple photosynthetic bacteria. Suppression of the change in the content of natural amino acids in the crushed material may mean that, when comparing the content of a specific natural amino acid relative to the total amino acids in marine purple photosynthetic bacteria with the content of the same specific natural amino acid relative to the total amino acids in the crushed material, the content of the specific natural amino acid relative to the total amino acids has not changed, or has changed only slightly. The change in the content of natural amino acids may be a value obtained by (content of the specific natural amino acid in the crushed material) / (content of the specific natural amino acid in marine purple photosynthetic bacteria). Preferably, (content of the specific natural amino acid in the crushed material) / (content of the specific natural amino acid in marine purple photosynthetic bacteria) is 0.05 to 4.0, more preferably 0.1 to 3.5, and even more preferably 0.2 to 3.0.

[0031] This section explains how the change in the total amount of amino acids and / or total free amino acids contained in the crushed material is suppressed compared to the total amount of amino acids and / or total free amino acids contained in marine purple photosynthetic bacteria. Suppression of the change in the total amount of amino acids and / or total free amino acids contained in the crushed material means that, when comparing the total amount of amino acids and / or total free amino acids contained in marine purple photosynthetic bacteria with the total amount of amino acids and / or total free amino acids contained in the crushed material, the total amount of amino acids and / or total free amino acids remains unchanged or changes only slightly. The amount of amino acids contained in the crushed material is preferably 85% by mass or more, more preferably 88% by mass or more, and even more preferably 90% by mass or more, relative to the total amount of amino acids contained in marine purple photosynthetic bacteria. The amount of free amino acids contained in the crushed material is preferably 85% by mass or more, more preferably 88% by mass or more, and even more preferably 90% by mass or more, relative to the total amount of free amino acids contained in marine purple photosynthetic bacteria.

[0032] Also, by the method for disrupting marine photosynthetic bacteria of the present embodiment, the change from organic nitrogen to inorganic nitrogen contained in the marine photosynthetic bacteria may be suppressed. The suppression of the change from organic nitrogen to inorganic nitrogen contained in the marine photosynthetic bacteria means that when comparing the content of total inorganic nitrogen contained in the marine photosynthetic bacteria with the content of total inorganic nitrogen contained in the disrupted product, the content of total inorganic nitrogen has not changed or the change is small. Also, the inorganic nitrogen may be contained as ammonia nitrogen or nitrate nitrogen.

[0033] It is preferable that the increase in the amount of inorganic nitrogen contained in the disrupted product is 50% or less, more preferably 47% or less, and even more preferably 44% or less, relative to the amount of amino acids contained in the marine photosynthetic bacteria. It is preferable that the amount of ammonia nitrogen contained in the disrupted product is 40% or less, more preferably 38% or less, and even more preferably 35% or less, relative to the amount of ammonia nitrogen contained in the marine photosynthetic bacteria. It is preferable that the amount of nitrate nitrogen contained in the disrupted product is 80% or less, more preferably 75% or less, and even more preferably 70% or less, relative to the amount of nitrate nitrogen contained in the marine photosynthetic bacteria. The inorganic nitrogen content in the marine photosynthetic bacteria and the disrupted product can be determined by measurement using the dry combustion method.

[0034] The method for disrupting marine purple photosynthetic bacteria according to this embodiment can suppress changes in the composition of carotenoids in the marine purple photosynthetic bacteria. The carotenoids contained in the marine purple photosynthetic bacteria constitute the carotenoids contained in the disrupted material, so the composition of carotenoids remains unchanged before and after disrupting the marine purple photosynthetic bacteria, and changes in the composition of carotenoids in the marine purple photosynthetic bacteria are suppressed. Alternatively, the amount of carotenoids contained in the marine purple photosynthetic bacteria may directly correspond to the amount of carotenoids contained in the disrupted material. The indicator of the change in the composition of carotenoids in marine purple photosynthetic bacteria may be the proportion of each carotenoid and / or the amount of each carotenoid. Changes in the carotenoid composition of marine purple photosynthetic bacteria may be, for example, (1) changes in the ratio of the content of each carotenoid in the crushed material compared with the ratio of the content of each carotenoid in the marine purple photosynthetic bacteria, or (2) changes in the total carotenoid content in the crushed material compared with the total carotenoid content in the marine purple photosynthetic bacteria.

[0035] Further, suppressing the change in the composition of carotenoids in marine photosynthetic bacteria may mean that the change in the composition of carotenoids contained in the crushed material is suppressed as compared with the composition of carotenoids contained in marine photosynthetic bacteria. Further, suppressing the change in the composition of carotenoids in marine photosynthetic bacteria may mean that when comparing the composition of carotenoids contained in the marine photosynthetic bacteria before being crushed by the method for crushing marine photosynthetic bacteria of the present embodiment with the composition of carotenoids contained in the crushed material, the composition of carotenoids has not changed or the change is small. The composition of carotenoids in the marine photosynthetic bacteria before being crushed by the method for crushing marine photosynthetic bacteria of the present embodiment may be determined by analyzing the composition of carotenoids in freeze-dried marine photosynthetic bacteria. Freeze-drying can provide a sample capable of measuring carotenoids in marine photosynthetic bacteria without affecting the composition of carotenoids contained in the marine photosynthetic bacteria. As demonstrated in the examples described later, the method for crushing marine photosynthetic bacteria of the present embodiment can suppress the change in the composition of carotenoids in the above-mentioned meaning, similarly to high-pressure crushing and ultrasonic crushing that can be conventionally used as methods for crushing bacteria, or in comparison with high-pressure crushing and ultrasonic crushing.

[0036] In the present specification, carotenoids mean a group of natural pigments showing yellow, orange, or red. In the present embodiment, the carotenoids are preferably carotenoids produced by marine photosynthetic bacteria. The carotenoids produced by marine photosynthetic bacteria are preferably carotenoids related to the spheroidenone pathway, and more preferably one or more selected from the group consisting of spheroidenone, spheroiden, dimethylspheroidenone, 3,4-dihydroxyspheroidenone, and neurosporene.

[0037] The composition of carotenoids contained in marine purple photosynthetic bacteria before or in the crushed material before crushing by the marine purple photosynthetic bacteria crushing method can be confirmed, for example, by analyzing the marine purple photosynthetic bacteria before or in the crushed material by ultraviolet-visible spectroscopy and mass spectrometry. More specifically, for example, as described in the examples, the composition of carotenoids can be measured by preparing the freeze-dried marine purple photosynthetic bacteria before crushing by the marine purple photosynthetic bacteria crushing method, or the crushed material, and then subjecting the prepared materials to ultraviolet-visible spectroscopy and mass spectrometry and comparing them with known carotenoid data. Here, for the viewpoint of measurement efficiency, the composition of carotenoids contained in the crushed material may be measured using dried crushed material obtained by the drying process of the crushed material described later.

[0038] Regarding the suppression of changes in the composition of carotenoids in marine purple photosynthetic bacteria, we will explain that the change in the ratio of each carotenoid contained in the crushed material is suppressed, compared to the ratio of each carotenoid contained in marine purple photosynthetic bacteria. The ratio of each carotenoid can also be rephrased as the ratio of the contents of different carotenoids.

[0039] Suppression of changes in the ratio of each carotenoid content in the crushed material may mean that, when comparing the ratio of each carotenoid content to the total carotenoids contained in marine purple photosynthetic bacteria with the ratio of each carotenoid content to the total carotenoids contained in the crushed material, the ratio of each carotenoid content to the total carotenoids has not changed, or has changed only slightly. The ratio of a specific carotenoid content to the total carotenoids contained in marine purple photosynthetic bacteria refers to the ratio when comparing the content of two or more carotenoids contained in marine purple photosynthetic bacteria. The ratio of each carotenoid content to the total carotenoids contained in the crushed material refers to the ratio when comparing the content of two or more carotenoids contained in the crushed material.

[0040] The change in the ratio of each carotenoid content may be calculated as follows: (ratio of each carotenoid content to the total carotenoids contained in the crushed material) / (ratio of each carotenoid content to the total carotenoids contained in marine purple photosynthetic bacteria).

[0041] This section explains how the change in the carotenoid content in the crushed material is suppressed compared to the carotenoid content in marine purple photosynthetic bacteria. Suppression of the change in the carotenoid content in the crushed material may mean that, when comparing the content of a specific carotenoid relative to the total carotenoids in marine purple photosynthetic bacteria with the content of that specific carotenoid relative to the total carotenoids in the crushed material, the content of that specific carotenoid relative to the total carotenoids has not changed, or has changed only slightly. The change in carotenoid content may be a value obtained by (content of the specific carotenoid in the crushed material) / (content of that specific carotenoid in marine purple photosynthetic bacteria). Preferably, (content of the specific carotenoid in the crushed material) / (content of that specific carotenoid in marine purple photosynthetic bacteria) is 0.05 to 4.0, more preferably 0.1 to 3.5, and even more preferably 0.5 to 3.0.

[0042] This section explains how the change in the total amount of carotenoids contained in the crushed material is suppressed compared to the total amount of carotenoids contained in marine purple photosynthetic bacteria. Suppression of the change in the total amount of carotenoids contained in the crushed material means that, when comparing the total amount of carotenoids contained in marine purple photosynthetic bacteria with the total amount of carotenoids contained in the crushed material, the total amount of carotenoids remains unchanged or changes only slightly. Preferably, the total amount of carotenoids contained in the crushed material is 90% by mass or more, more preferably 93% by mass or more, and even more preferably 95% by mass or more, relative to the total amount of carotenoids contained in marine purple photosynthetic bacteria.

[0043] (Step of drying the crushed material) The method for crushing marine purple photosynthetic bacteria of this embodiment may include a step of drying the crushed material. Here, the crushed material may be the same as that described above. The step of drying the crushed material is carried out after the step of obtaining the crushed material of marine purple photosynthetic bacteria. Furthermore, the step of drying the crushed material can be used to obtain dried crushed material. In addition, the drying step of the crushed material usually does not change the composition of amino acids and carotenoids contained in the crushed material.

[0044] Methods for drying the crushed material include, for example, forced-air drying using a blower or the like to apply hot or cold air; airless drying using heat to evaporate moisture; spray drying in which the slurry is suspended in a suitable buffer and then the suspension is sprayed into a gas for rapid drying; freeze-drying; vacuum drying using a vacuum pump or the like to remove air in a sealed container; air drying (including sun drying) by leaving it exposed to the outside air; and combinations thereof. Air drying is preferred from the viewpoint of suppressing changes in the amino acid composition contained in marine purple photosynthetic bacteria.

[0045] The temperature at which the crushed material is dried is not particularly limited, but is preferably 10°C to 55°C, more preferably 15°C to 40°C, and even more preferably 20°C to 30°C.

[0046] The drying time for the crushed material is not particularly limited as long as it is the time required for the acetone solvent used in the process of obtaining the crushed marine purple photosynthetic bacteria to be removed, but it is preferably 30 minutes to 5 days, more preferably 1 hour to 4 days, and even more preferably 1.5 hours to 3 days. Furthermore, when drying crushed material with a thickness of 1 mm, it is preferable to dry it in a ventilated hood for 2 to 3 days, or in a dryer at 30°C for 3 to 4 hours.

[0047] Furthermore, the dried crushed material may be a liquid, a solid, or a mixture thereof, but it is preferably a solid, and more preferably a powder.

[0048] (Step of culturing marine purple photosynthetic bacteria) As described above, the method for crushing marine purple photosynthetic bacteria of this embodiment may include a step of culturing marine purple photosynthetic bacteria. It is preferable that the step of culturing marine purple photosynthetic bacteria be carried out before the step of obtaining the crushed product of marine purple photosynthetic bacteria.

[0049] The process of culturing marine purple photosynthetic bacteria may result in marine purple photosynthetic bacteria having a high amino acid content. In this specification, culturing means the process of increasing the number of bacteria and accumulating nutrients such as amino acids within the bacterial cells by culturing bacteria under specific conditions.

[0050] The process of culturing marine purple photosynthetic bacteria may result in marine purple photosynthetic bacteria having a high carotenoid content. In this specification, culturing means the process of increasing the number of bacterial cells and accumulating nutrients such as carotenoids within the bacterial cells by culturing the bacteria.

[0051] As for the cultivation method, methods known as mass culture methods can be employed, including continuous culture methods and batch culture methods. The cultivation of the inoculum and the marine purple photosynthetic bacteria can be carried out as appropriate and are not particularly limited, but it is preferable that the marine purple photosynthetic bacteria are grown by culturing them.

[0052] The cultivation of marine purple photosynthetic bacteria may be carried out under the irradiation of near-infrared light, which marine purple photosynthetic bacteria use for photoautotrophic growth, or under far-red light. The far-red light may have a peak wavelength range of 700 nm to 860 nm. The method of far-red light irradiation is not particularly limited; conventional irradiation methods used for culturing marine purple photosynthetic bacteria may be used.

[0053] The culture time should be sufficient to allow marine purple photosynthetic bacteria to accumulate biomolecules such as amino acids and carotenoids, and the culture temperature may be set appropriately according to the optimal culture temperature of marine purple photosynthetic bacteria. The culture temperature is not particularly limited, but it is preferably between 20°C and 40°C. Regarding the culture time, for example, culture may be carried out until the intermediate logarithmic growth phase is reached, and then the culture medium may be changed and culture may be continued until the stationary phase is reached.

[0054] Culturing can be carried out under suitable atmospheric conditions, but performing it under nitrogen-containing conditions allows for the fixation of atmospheric nitrogen, enabling the production of nitrogen-rich crushed materials such as amino acids without the need to add a nitrogen source to the culture medium. The nitrogen concentration in the culture medium can also be increased by bubbling nitrogen gas into it.

[0055] The culture medium used is not particularly limited as long as it is a medium capable of culturing marine purple photosynthetic bacteria, but conventionally known growth media may be used. The culture medium is not particularly limited, but for example, natural seawater may be used, or a seawater-based medium utilizing natural seawater may be used. The growth medium may contain an organic carbon source or an inorganic carbon source. If an inorganic carbon source is included, the culture medium does not need to contain an organic carbon source. In this embodiment, an inorganic carbon source is preferably used, and carbon fixation may be promoted by culturing in a medium in which an organic carbon source is not used in some cases. As the culture medium used, for example, the marine broth medium described in the examples is preferred.

[0056] (Step of collecting marine purple photosynthetic bacteria) As described above, the method for crushing marine purple photosynthetic bacteria of this embodiment may include a step of collecting marine purple photosynthetic bacteria. Preferably, the step of collecting marine purple photosynthetic bacteria is performed after the step of culturing marine purple photosynthetic bacteria and before the step of obtaining crushed marine purple photosynthetic bacteria.

[0057] The process of collecting marine purple photosynthetic bacteria may also mean the process of removing components derived from the culture medium (e.g., culture medium, amino acids, organic carbon sources, and inorganic carbon sources, etc.) and obtaining only the bacteria from the culture medium. When components derived from the culture medium are removed in the process of collecting marine purple photosynthetic bacteria, the components contained in the crushed material are components derived from the collected marine purple photosynthetic bacteria themselves.

[0058] The step of collecting marine purple photosynthetic bacteria may be carried out by washing and recovering the cultured marine purple photosynthetic bacteria. In the collection step, washing may be performed first, then recovery and collection, or recovery may be performed first and then washing. In the case of collection, collection may be performed from one or more culture tanks. Recovery of marine purple photosynthetic bacteria can be carried out by conventionally known methods for recovering bacteria from the culture medium, but for example, centrifugation, standing, or a combination thereof is preferred. Washing of marine purple photosynthetic bacteria is preferably done by, for example, suspending the recovered marine purple photosynthetic bacteria in a desired solution and washing, or by washing while desalting using conventionally known methods such as ultrafiltration. The number of times recovery and / or washing for collection of marine purple photosynthetic bacteria is not particularly limited, but it may be done once or more.

[0059] The process of collecting marine purple photosynthetic bacteria is preferably carried out by the following methods, for example, from the viewpoint of obtaining only bacteria from the culture medium while removing components derived from the culture medium: The culture medium containing marine purple photosynthetic bacteria is precipitated by centrifugation or standing, and the supernatant containing components derived from the culture medium is removed. The precipitated marine purple photosynthetic bacteria can be suspended in water, and the suspension can be precipitated by centrifugation or standing, and the supernatant containing salts and components derived from the culture medium can be removed. Alternatively, salts and components derived from the culture medium can be removed by ultrafiltration of the culture medium containing marine purple photosynthetic bacteria, or the suspension of precipitated marine purple photosynthetic bacteria suspended in water.

[0060] (Other steps) The method for crushing marine purple photosynthetic bacteria of this embodiment may further include one or more steps from extraction, desalting, granulation, sizing, and acid / alkali treatment, in addition to the step of obtaining crushed marine purple photosynthetic bacteria. The desalting step may be included for the purpose of removing salt present in the culture solution or washing solution, and the acid / alkali treatment step may be included for the purpose of adjusting the pH of the culture solution or washing solution. Furthermore, the steps of extraction, desalting, granulation, sizing, and acid / alkali treatment may be carried out by conventionally known methods. In addition, the method may include a step of freeze-drying the collected marine purple photosynthetic bacteria, and the marine purple photosynthetic bacteria stored at low temperature after freeze-drying may be used in the step of obtaining crushed marine purple photosynthetic bacteria.

[0061] (Uses of the crushed material) In this embodiment, the crushed material is preferably used as a nutrient. Here, the crushed material may be the same as that described above. Furthermore, it is preferable that the crushed material be used as a nutrient after undergoing the process of obtaining crushed marine purple photosynthetic bacteria and the subsequent process of drying the crushed material. The method for crushing marine purple photosynthetic bacteria in this embodiment is a method that suppresses changes in the amino acid composition of marine purple photosynthetic bacteria when the marine purple photosynthetic bacteria are crushed. Therefore, the crushed material obtained by the method for crushing marine purple photosynthetic bacteria in this embodiment has a high amino acid content. For this reason, the crushed material can be used as a nutrient.

[0062] In this embodiment, the term "nutrient solution" may refer to a substance used to supplement nutrients, and its dosage form, degree of purification, etc., are not particularly limited. As described above, the crushed material obtained by the crushing method of marine purple photosynthetic bacteria in this embodiment has a high amino acid content, so by using the crushed material, a nutrient solution containing a large amount of amino acids can be obtained. The crushed material may be used as a nutrient solution on its own, or it may be processed appropriately with other components as needed to form a nutrient solution. The nutrient solution may be used in mixture with conventionally known nutrients. Furthermore, the dosage form of the nutrient solution can be appropriately selected according to the intended use of the nutrient solution.

[0063] Uses of nutritional supplements include, for example, fertilizer, animal feed, food, and culture medium. Examples of fertilizers include those used for agricultural crops (e.g., grains, vegetables, fruits, flowers, legumes, and tea). Examples of animal feed include those used for livestock (e.g., cattle, pigs, chickens, horses, sheep, and goats), pets (dogs, cats, hamsters, rabbits, parakeets, reptiles, amphibians, and insects), and aquaculture fish and shellfish (e.g., fish, shellfish, and crustaceans). Examples of food include foods manufactured by adding nutritional supplements to food ingredients and adding other food additives as appropriate, according to known methods depending on the type of food. Examples of food include general foods, health foods, functional foods, nutritional supplements, and health functional foods (e.g., foods for specified health uses, foods with functional claims, and nutrient function foods), and it is preferable that the crushed material has a high amino acid content, making it a high-protein food. Examples of culture media include those for culturing cells, microorganisms, and bacteria. From the viewpoint of the crushed material having a high amino acid content, it is preferable to use it as a culture medium for culturing cells for cultured meat. Furthermore, in this embodiment, since the crushed material contains carotenoids, it can also be used as a color enhancer. Examples of color enhancers containing the crushed material include fish and shellfish such as goldfish, carp, salmon, sea bream, rainbow trout, yellowtail, amberjack, shrimp, and crabs, birds such as chickens and parakeets, other livestock animals, and plants and animals.

[0064] (Method for suppressing changes in amino acid composition or carotenoid composition) The method for crushing marine purple photosynthetic bacteria of this embodiment can suppress changes in the amino acid composition of marine purple photosynthetic bacteria. That is, one aspect of this embodiment may be a method for suppressing changes in the amino acid composition of marine purple photosynthetic bacteria, which includes the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria.

[0065] The method for disrupting marine purple photosynthetic bacteria according to this embodiment can suppress changes in the composition of carotenoids in marine purple photosynthetic bacteria. That is, one aspect of this embodiment may be a method for suppressing changes in the composition of carotenoids in marine purple photosynthetic bacteria, which includes the step of disrupting marine purple photosynthetic bacteria in an acetone solvent to obtain a disrupted product of marine purple photosynthetic bacteria.

[0066] One aspect of this embodiment may be a method for suppressing changes in the amino acid composition or carotenoid composition of marine purple photosynthetic bacteria, which includes the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria, and it is preferable that the method suppresses changes in the amino acid composition and carotenoid composition of marine purple photosynthetic bacteria.

[0067] The method for suppressing changes in the amino acid composition or carotenoid composition of marine purple photosynthetic bacteria according to this embodiment includes the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria. The method for suppressing changes in the amino acid composition or carotenoid composition of marine purple photosynthetic bacteria according to this embodiment may further include one or more steps selected from the group consisting of a step of drying the crushed product, a step of culturing marine purple photosynthetic bacteria, a step of collecting marine purple photosynthetic bacteria, and other steps. The description of these steps is the same as above.

[0068] Some embodiments of the above-described embodiments of the present invention are described below. The present invention will be specifically explained below with reference to these embodiments, but the present invention is not limited to these embodiments.

[0069] [Example 1] Comparison of total nitrogen (N), total carbon (C), and inorganic nitrogen depending on the crushing method [Example 1-1: Acetone crushing vs. high-pressure crushing] 1. R. Culture and Recovery of sulfidophyllum: Rhodovulum sulfidophyllum (DSM 1374; W4, LMG 5202) (American Type Culture Collection, ATCC) (R. sulfidophyllum), a marine purple photosynthetic non-sulfur bacterium, was cultured in marine broth medium (Merck, Millipore Sigma, Massachusetts, United States) in a 2 L Schott bottle under continuous far-red light irradiation (730 nm, 45 W / m²) at room temperature. ２ The cells were cultured with stirring (350 rpm) using (Y104-R660 / W40 / IR73-31W-EI0U1LW-010, YUMEX Solutions). After 5 days of culture, the cells were collected by centrifugation (7900 rpm, 10 minutes), and the cells were resuspended in pure water to remove salt. The washed cells were then collected by centrifugation (7900 rpm, 30 minutes) and stored at -80°C until use.

[0070] 2. Cell lysation and drying The cells from (1) to (3) below (each tested in 3 batches) were divided equally into 3 fractions. (1) High-pressure lysation and freeze-drying (PANDA + lyophilization) The collected and washed cells were completely resuspended in pure water (1 g CFW (Cell fresh weight): 5 ml water). The cell suspension was treated 7 times under 1000 bar conditions in a high-pressure homogenizer (Panda PLUS 2000, GEA, Düsseldorf, Germany) to lysate the cells. The resulting cell lysates were freeze-dried (FDL-1000, EYELA). (2) Acetone disruption and air drying The collected and washed cells were stirred in acetone (1 g CFW: 1.5 ml acetone) at 700 rpm for 30 minutes to completely resuspend them. However, if the cells did not completely suspend in acetone, stirring was continued for 1 hour. The disrupted cells were spread on a tray and air-dried at room temperature for 2 to 3 days. (3) Undisrupted cells The collected and washed cells were freeze-dried without lysis. For (1) to (3) above, complete cell disruption was confirmed by plated 100 μL of the disrupted cells on Marine Broth agar and heated at 30°C under continuous irradiation of far-red light (730 nm, 20-30 Wm). －２ This was confirmed by culturing the cells in ) the cells.

[0071] [Example 1-2: High-pressure fracturing vs. ultrasonic fracturing] 1. Culture and recovery of R. sulfidophyllum Rhodovulum sulfidophyllum (DSM 1374; W4, LMG 5202) (ATCC), a marine purple photosynthetic non-sulfur bacterium, was cultured in a 2L Schott bottle in artificial seawater (Marine Standard, manufactured by Nippon Kaisui Co., Ltd., 3g / L) to which 0.1% yeast extract (Cat. No. A1202HA, Solabia Biokar Diagnostics) and 0.5% peptone (Cat. No. 24-0970, Sigma Aldrich) had been added, at room temperature and 45 W / m². ２The cells were cultured with stirring (350 rpm) using (Y104-R660 / W40 / IR73-31W-EI0U1LW-010, YUMEX Solutions). After 5 days of culture, the cells were collected by centrifugation (7900 rpm, 10 minutes) and resuspended in pure water to remove salt. The washed cells were then collected by centrifugation (7900 rpm, 30 minutes) and stored at -80°C until use.

[0072] 2. Cell lysation and drying The cells from (1) to (2) below (each tested in one batch) were divided equally into two fractions. (1) High-pressure lysation and freeze-drying (PANDA + lyophilization) The collected and washed cells were completely resuspended in pure water (1 g CFW: 5 ml water). The cell suspension was treated seven times under 1000 bar conditions in a high-pressure homogenizer (Panda PLUS 2000, GEA, Düsseldorf, Germany) to lyse the cells. The cell lysates were freeze-dried (FDL-1000, EYELA). (2) Ultrasonic disruption and lyophilization The collected and washed cells were ultrasonically disrupted at 19.2 kHz, 60% amplification, 35 rpm (3 cycles) (Mitsui Electric UX600, Chiba, Japan). The cell lysates were dried by lyophilization (FDL-1000, EYELA). To confirm that the cells were completely disrupted, 100 μL of the lysates was plated on Marine Broth agar and heated at 30°C under continuous irradiation with far-red light (730 nm, 20-30 Wm). －２ This was confirmed by culturing the cells in ) the cells.

[0073] [Example 1-3] Analysis of total nitrogen (N), total carbon (C), and inorganic nitrogen The total nitrogen (N) and total carbon (C) of the crushed bacterial-dried biomass were analyzed by dry combustion. Ammonia nitrogen (Ammonia-N) and nitrate nitrogen (Nitrate-N) were extracted with 1 M potassium chloride in a 1:10 ratio and then evaluated by spectrophotometric method (Vegetch Co., Ltd. and Chemical Analysis Center). The results for unfused cells, acetone crushing and air drying, and PANDA crushing and freeze-drying in Example 1-1 are shown in the table below. Values ​​are expressed as % dry cell weight (CDW).

[0074] The results of high-pressure crushing and freeze-drying (PANDA + lyophilization) and ultrasonic crushing and freeze-drying (Ultrasonication + lyophilization) in Examples 1-2 are shown in the table below. The values ​​are expressed as % dry cell mass (CDW, Cell Dry weight).

[0075] Acetone crushing resulted in a lower conversion rate of organic nitrogen to inorganic nitrogen compared to high-pressure crushing. Furthermore, the total amount of carbon did not change depending on the crushing method.

[0076] [Example 2] Amino acid analysis and preparation of analytical samples 1. Preparation of amino acid analysis samples Bacterial dried biomass derived from R. sulfidophyllum, processed as described in (1) or (2) below, was used for amino acid analysis. (1) High-pressure crushing and / or ultrasonic treatment, and freeze-drying ・AF1 was obtained using R. sulfidophyllum cultured in marine broth medium (MB) in the same manner as in Example 1-1, and by high-pressure crushing and freeze-drying in the same manner as in Example 1-1 (1). ・AF2 and AF3 were obtained using R. sulfidophyllum cultured in marine broth medium in the same manner as in Example 1-1, and by crushing and freeze-drying using a combination of high-pressure crushing and ultrasonic crushing in the same manner as in Examples 1-2 (1) and (2). AF4 was obtained by crushing and freeze-drying using a combination of high-pressure crushing and ultrasonic crushing, as in Example 1-2 (1) and (2), using R. sulfidophyllum cultured in marine broth medium and natural seawater (NSW) with 0.1% yeast extract and 0.5% peptone added, as in Example 1-2. (2) Acetone crushing and air drying AF5 and AF6 were obtained by crushing and air-drying using acetone crushing and air drying, as in Example 1-1 (2), using R. sulfidophyllum cultured in marine broth medium, as in Example 1-1. AF7 was obtained by crushing R. sulfidophyllum cultured in artificial seawater (Marine Standard, manufactured by Nippon Kaisui Co., Ltd., 3 g / L) with 0.1% yeast extract and 0.5% peptone added, as in Example 1-2. It was obtained by using sulfidophyllum and performing acetone crushing and air drying in the same manner as in Example 1-1(2).

[0077] 2. Analysis of Amino Acids At the Biomaterial Analysis Support Unit, Research Resources Department, RIKEN Brain Science Institute, an amino acid analyzer (L-8900, Hitachi) using post-columnar hydrin derivatization of amino acids was used to quantify free amino acids and total amino acids (excluding cysteine) of protein-constituting amino acids in dried cellular biomass. To quantify free amino acids, 5.3 mg of lyophilized sample was sonicated in 206 μL of 0.2 M perchloric acid for several minutes and incubated at 0°C for 30 minutes. After centrifugation at 12,000 g, the supernatant was diluted 3.3 times with lithium citrate buffer and used for analysis. To quantify total amino acids (amino acids constituting 19 proteins), 1.2 mg of lyophilized sample was hydrolyzed in 50 μL of 4 M methanesulfonic acid at 110°C for 20 hours. The volume of the hydrolysate was adjusted to 100 μL with ultrapure water and filtered through a 0.45 μm filter. The filtrate was diluted 20-fold with lithium citrate buffer and subjected to analysis. Approximately 20% of the total tryptophan content was destroyed during the acid hydrolysis procedure. The analysis results for AF1 to AF7 are shown in the table below. Table 5 shows the analysis results for the total amino acid content and total free amino acid content for AF1 to AF4, and Table 6 shows the analysis results for the total amino acid content and total free amino acid content for AF5 to AF7. Since AF1 to AF4 are estimated to represent the amino acid content contained in marine purple photosynthetic bacteria before disruption, the average values ​​for AF1 to AF4 and the average values ​​for AF5 to AF7 were compared. Differences in culture conditions did not affect the amino acid content contained in marine purple photosynthetic bacteria. As a result, the amount of amino acids in the disrupted material was 92.8% by mass of the total amount of amino acids contained in marine purple photosynthetic bacteria. Furthermore, the amount of free amino acids contained in the crushed material was 92.8% by mass of the total amount of free amino acids contained in the marine purple photosynthetic bacteria.

[0078] Table 7 shows the analysis results for AF1-AF4, and Table 8 shows the analysis results for all amino acids (19 amino acids that make up proteins) of AF5-AF7. Figure 1 shows the graph corresponding to Table 7, and Figure 2 shows the graph corresponding to Table 8. Since AF1-AF4 are estimated to represent the content of each amino acid in marine purple photosynthetic bacteria before disruption, the content of each amino acid in AF1-AF4 and the content of each amino acid in AF5-AF7 were compared. As a result, the content of each natural amino acid contained in AF1-AF4 and AF5-AF7 remained unchanged.

[0079] [Example 3] Confirmation of Cell Disruption The cells were confirmed to be disrupted according to the following method. If bacterial growth was observed, the cells were considered not to have been completely disrupted. 1. High-Pressure Disruption The cells obtained in Example 1-1 were completely resuspended in pure water (1 g CFW (Cell fresh weight): 5 ml water). Then, the cells were disrupted by processing them for a specified number of times at 180 ml / min under conditions of 80-100 MPa using a high-pressure homogenizer (PandaPLUS 2000, GEA, Düsseldorf, Germany) under conditions of 80-100 MPa. 100 μL of the disrupted material diluted to each concentration was spread onto Marine Broth agar medium and cultured for 3 days at 30°C under continuous irradiation of far-red light. The number of processing cycles using the high-pressure homogenizer and the bacterial growth status after culture are shown in Figure 3. It was found that the cells were completely disrupted by more than seven treatments with a high-pressure homogenizer. 2. Cells obtained in ultrasonic disruption Example 1-1 were ultrasonically disrupted at 19.2 kHz, 60% amplification, 70 rpm (2 cycles) (Mitsui Electric UX600, Chiba, Japan). 100 μL of the resulting cell disruption solution was spread onto Marine Broth agar medium and heated at 30°C under continuous irradiation with far-red light (20-30 Wm). －２)(2) to 5 days. The growth status of the bacteria after culture is shown in FIG. 4. It was found that the cells were completely disrupted by ultrasonic disruption. 3. Acetone disruption The cells obtained in Example 1-1 were completely resuspended in acetone (1 g CFW: 1.5 ml acetone). The suspension was stirred at 100 rpm for 1 hour at room temperature. 100 μL of the obtained cell disruption solution was applied to Marine Broth agar medium, and at 30 ° C., under continuous irradiation with far-red light (20 - 30 Wm －２ )(2) to 5 days. The growth status of the bacteria after culture is shown in FIG. 5. It was found that the cells were completely disrupted by acetone disruption. It was found that the disruption efficiency was high compared to high-pressure disruption and ultrasonic disruption.

[0080] [Example 4] Nitrogen (N), phosphoric acid (P ２ O ５ ), potassium oxide (K ２ O), carbon (C), and analysis of inorganic nitrogen [Example 4-1: High-pressure disruption vs ultrasonic disruption] 1. R. sulfidophilum culture and recovery Rhodovulum sulfidophilum (DSM 1374; W4, LMG 5202) (ATCC), a marine red photosynthetic non-sulfur bacterium, in 2 L Schott bottles containing 0.1% yeast extract (Cat. No. A1202HA, Solabia Biokar Diagnostics) and 0.5% peptone (Cat. No. 24-0970, Sigma Aldrich) added to artificial seawater (Marine Standard, Nippon Suisui Co., Ltd., 3 g / L), at room temperature, 45 W / m ２ (Y104-R660 / W40 / IR73-31W-EI0U1LW-010, YUMEX Solutions) with stirring (350 rpm). The preculture was inoculated with a single colony on marine agar into 50 mL of MB and cultured for 5 days at 25 Wm －２ under continuous far-red light (730 nm). In the main culture, (0.5% v / v) of the 100 mL or 500 mL preculture was inoculated into 2 L or 10 L of fresh medium. After culturing for 5 days, the cells were recovered by centrifugation (13,000 xg, 10 minutes) and resuspended in pure water to remove salts. The 10 L scale culture was 7.5 mgL －１Chitosan 100 (Fujifilm Wako, Osaka, Japan) was dissolved in 0.01% acetic acid (Fujifilm Wako) and allowed to aggregate for 2-3 days. The cells were then centrifuged and resuspended in water. After washing, the cells were recovered by an additional centrifugation step (13,000 x g, 30 minutes) and stored at -80°C until use.

[0081] 2. Cell lysis and drying The cells from (1) to (2) below (each tested in 3 batches) were divided equally into 3 fractions. (1) High-pressure lysis and freeze-drying (PANDA + lyophilization) The collected and washed cells were completely resuspended in pure water (1 g CFW: 5 ml water). The cell suspension was homogenized in a high-pressure homogenizer (PandaPLUS 2000, GEA, Düsseldorf, Germany) at 80-100 MPa, 180 ml min. －１ Under the specified conditions, the cells were treated seven times and lysed. The lysates were dried by freeze-drying (FDL-1000, EYELA). (2) Acetone lysation and air drying The collected and washed cells were completely resuspended in acetone (1g CFW:1ml acetone) at 500-600 rpm for 30 minutes to 1 hour. The lysates were spread on a tray and air-dried at room temperature for 48-72 hours. (3) Unlysed cells The collected and washed cells were freeze-dried without lysis. For (1) to (3) above, complete cell lysation was confirmed by plated lysates on Marine Broth agar and irradiated with far-red light at 30°C (730nm, 20-30Wm). －２ The results were confirmed by culturing the cells for 5 days.

[0082] 3. Cell Disruption and Drying The cells from (1) to (2) below (each tested in one batch) were divided equally into two fractions. (1) High-pressure disruption and lyophilization (PANDA + lyophilization) The collected and washed cells were completely resuspended in pure water (1 g CFW: 5 ml water). The cell suspension was treated as described in 2. above. (2) Ultrasonic disruption and lyophilization (Ultrasonication + lyophilization) The collected and washed cells were ultrasonically disrupted at 19.2 kHz, 60% amplification, 35 rpm (3 cycles) (Mitsui Electric UX600, Chiba, Japan). The cell lysates were dried by lyophilization (FDL-1000, EYELA). Regarding (1) and (2) above, complete cell disruption can be confirmed by platedting the disrupted cells onto Marine Broth agar and irradiating them at 30°C under continuous far-red light (730 nm, 20-30 Wm). －２ The results were confirmed by culturing the cells for 5 days.

[0083] 4. Comparison of Total Inorganic Nitrogen Content Ammonia nitrogen (Ammonia-N) and nitrate nitrogen (Nitrate-N) of crushed bacterial-dried biomass were extracted with 1 M potassium chloride in a 1:10 ratio and evaluated by spectrophotometric analysis (Vegetch Co., Ltd. and Chemical Analysis Center). Total nitrogen (N) of crushed bacterial-dried biomass was analyzed by dry combustion. The results are shown in Figure 6. Data were obtained from three independent 2 L cultures (n=3), and the mean ± standard error (SEM) is shown. Asterisks indicate significant differences between methods tested by two-way ANOVA (Tukey test). * p < 0.05, ** p < 0.01, *** p < 0.001. The nitrate nitrogen content of cells disrupted by a high-pressure homogenizer was significantly higher compared to cells disrupted by acetone and undisrupted cells. This was also reflected in the total inorganic nitrogen content, which was similar between undisrupted and acetone-disrupted cells. It should be noted that ammonium nitrogen is inherently unstable and is rapidly converted to nitrate by nitrifying bacteria, so it is possible that it was introduced into the non-sterile dissolution system or the high-pressure homogenizer workflow. In addition, it is possible that the prolonged processing time led to enzyme-dependent degradation of arginine or glutamate, increasing the nitrate nitrogen concentration in the high-pressure-disrupted cells.

[0084] [Example 4-2: Nitrogen (N), phosphoric acid (P ２ O ５ ), potassium oxide (K ２ [Analysis of O)] The total nitrogen (N) of the crushed bacterial-dried biomass obtained in Example 4-1 was analyzed by dry combustion. In addition, the phosphate (P) of the crushed bacterial-dried biomass obtained in Example 4-1 was analyzed. ２ O ５ ), potassium oxide (K ２The analysis of O) was evaluated according to the method described in Reference 1. Reference 1: Morey-Yagi, SR et al. Utilization of lysed and dried bacterial biomass from the marine purple photosynthetic bacterium Rhodovulum sulfidophilum as a sustainable nitrogen fertilizer for plant production. npj Sustainable Agriculture 2, (2024). The results for unlysed cells, acetone lysed and air-dried cells, and high-pressure lysed and freeze-dried cells are shown in the table below. The values ​​are expressed as % dry cell mass (CDW, Cell Dry weight). The data represent the mean ± standard error (SEM) and were obtained from three independent 10L cultures (n=3). Two-way ANOVA (Tukey test) did not reveal any statistically significant differences between the treatments.

[0085] [Example 5] Analysis of Amino Acids Based on Different Crushing Methods The crushed bacterial-dried biomass obtained in Example 4-1 was divided equally into three fractions. The free and total amino acid content, excluding cysteine ​​and tryptophan, which are protein-derived amino acids, was measured using an amino acid analyzer (L-8900, Hitachi) and analyzed using post-columnar hydrin derivatization of amino acids, provided by the Biomaterial Analysis Support Unit, Research Resources Department, RIKEN Brain Science Institute. To quantify free amino acids, 5 mg of air-dried sample was sonicated with 200 μL of 0.2 M perchloric acid for approximately 2 minutes, and then incubated at 0°C for 30 minutes. After centrifugation at 12,000 x g for 5 minutes, the supernatant was diluted 3.3 times with lithium citrate buffer (pH 2.2) (123-02505, Fujifilm Wako Pure Chemical Corporation), and 90 μL was used for analysis. To quantify all amino acids (18 of the 20 amino acids necessary for protein formation, excluding cysteine ​​and tryptophan), 1 mg of freeze-dried sample was hydrolyzed in 100 μL of 6 M hydrochloric acid at 110°C for 20 hours. The hydrolysate was adjusted to 100 μL with ultrapure water and filtered through a 0.45 μm filter. The filtrate was diluted 20-fold with lithium citrate buffer, and 15 μL was used for analysis. The results are shown in Figure 7. Data were obtained from three independent cultures (n=3). Data represent mean ± standard error (SEM). Asterisks indicate significant differences between the disrupted group and the undisrupted control group using two-way ANOVA (Tukey test). * p < 0.05, ** p < 0.01, *** p < 0.001.

[0086] [Example 6] Analysis of Carotenoids [Example 6-1] 1. Extraction and Analysis of Carotenoids Unless otherwise specified, the photochromic pigment was extracted from R. sulfidophyllum obtained in Example 4-1 using chloroform:methanol (1:1, v / v). Approximately 20 mL of the solvent mixture was added to 3-5 mg of the dried bacterial material and mixed uniformly. Extraction was repeated until the pellet became colorless. The bound extract (approximately 50-100 mL) was evaporated under reduced pressure, and the dried residue was redissolved in an appropriate amount of solvent and analyzed with a spectrophotometer. The absorbance spectrum was measured using a spectrophotometer (Hitachi, L-6000). The sample was diluted 5-10 times as needed to bring the absorbance within a measurable range. The concentrations of carotenoids and bacteriochlorophyll a (BChla) were calculated using the specific extinction coefficients of 2785 and 1101, respectively, for spheroidenone and BChla. The extracts were analyzed using a Waters Xevo G2-S QTOF mass spectrometer with an Acquity UPLC system (Waters Corporation, Milford, MA, USA) and a BEH C18 column (1.7 μm, 2.1 × 100 mm) using MeCN:H ２ Analysis was performed using a 15-minute linear gradient from O (85:15) to MeCN:MeOH (65:35) (flow rate 0.4 mL / min). UV-VIS spectra were recorded in the range of 200 to 600 nm using a photodiode array detector (PDA). Mass spectra were acquired in the range of m / z 100–1500 using electrospray ionization (ESI) with a capillary voltage of 3.2 kV, a cone voltage of 20 eV, and a source temperature of 120 °C. Nitrogen was used as the nebulizing gas at a flow rate of 30 L / h. MS / MS spectra were acquired using a quadrupole-TOF apparatus with an impact energy of 20 V and argon as the collision gas. Carotenoids were identified by comparing the retention times of UV-VIS, MS, and MS / MS spectral data with those of standards. Carotenoid extraction and analysis were performed by SEIKEN Institute of Production Development (Kyoto, Japan).

[0087] 2. Comparison of Carotenoid Profiles by Acetone and Ultrasonic Disruption Considering the lipophilicity of acetone and the possibility of degradation of lipophilic pigments during processing, the pigment content in Processed Biomass (PB) was tested using both high-pressure and acetone disruption methods and compared with undisrupted cells. The results are shown in the table below. Data represent the mean ± standard error (SEM) and were obtained from three independent 10L cultures (n=3). The statistical significance of the difference between the disrupted group and the undisrupted control group was shown to be ***p < 0.001 using two-way ANOVA (Tukey test). There was no significant difference in total carotenoid content between the acetone-disrupted and high-pressure-disrupted groups. The carotenoids in R. sulfidophyllum were consistent with references 2 and 3, consisting of components of the spheroidene pathway. Specifically, spheroidenone, spheroidene, dimethylspheroidenone, and others such as 3,4-dihydroxyspheroidenone and neurosporene were identified, with spheroidenone being the most abundant (approximately 83% of the total carotenoid content). Both acetone-crushed and high-pressure-crushed PB showed similar total and individual carotenoid content to the uncrushed control. However, crushing treatment caused degradation of bacteriochlorophyll, which was thought to be due to prolonged treatment time and exposure to light. Reference 2: Maeda, I. et al. Unusual Accumulation of Demethylspheroidene in Anaerobic-Phototrophic Growth of crtA-Deleted Mutants of Rhodovulum sulfidophilum. Curr Microbiol 51, 193-197 (2005). Reference 3: Distribution and Biosynthesis of Carotenoids. in Advances in Photosynthesis and Respiration 97-117 (Springer Netherlands, Dordrecht, 2009). doi:10.1007 / 978-1-4020-8815-5_6.

[0088] [Example 6-2] 1. Evaluation of pigments extracted during acetone lysation The cell pellet (100 g or more of FW) obtained in Example 4-1 was manually resuspended in acetone at a ratio of 1:1 (w / v) or 1:1.5 (w / v) until no visible clumps remained. The suspension was stirred with a magnetic stirrer. Aliquots (1 mL) were sampled at 0 minutes, 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, and 2.5 hours after manual resuspending, centrifuged at 9,000 x g for 5 minutes, and the supernatant was diluted 20-fold for UV-Vis spectroscopy. To identify the corresponding carotenoid peaks shown in Figure 8a, acetone-lysed cells were re-extracted with acetone:methanol (7:2), centrifuged at 9,000 x g for 5 minutes, and the supernatant was used to obtain a UV-Vis spectrum. To quantify the pigments released during acetone lysis (Figure 8b), cell pellets from approximately 10 mL of cell culture medium (each weight recorded) were lysed in acetone for 30 minutes. The UV-Vis spectrum of the supernatant was recorded, and the concentrations of carotenoids and bacteriochlorophyll a (BChla) were calculated using the specific extinction coefficients of 2785 and 1101 for spheroidenone and BChla, respectively. To reduce scattering effects, the diluted supernatant was centrifuged at 9000 x g for 5 minutes before measurement (Figure 9a). The total carotenoid and bacteriochlorophyll a content was obtained by treating 500 mL of the same culture medium with a similar weight of lyophilized, un-ruptured biomass as a cell pellet, using trichloromethane:methanol (1:1, v / v).

[0089] 2. Extraction and Analysis Results of Carotenoids To evaluate whether the acetone used for cell disruption was reusable, the dyes dissolved in the acetone during cell disruption were analyzed. The supernatant after acetone disruption (CFW:acetone = 1:1) showed absorption spectral peaks corresponding to bacteriochlorophyll a (BChla) at 358 nm, 575 nm, and 766 nm, representing the Soret band, Qx band, and Qy band, respectively (Figure 8a). To identify the peak range of carotenoids, the pellet after acetone disruption was partially dissolved in an acetone:methanol mixture, and an absorption spectrum was obtained at 483 nm, which corresponds to spheroidenone (Figure 8a). The absence of a peak at approximately 480 nm in the supernatant absorption spectrum suggested that carotenoids were retained during cell disruption. However, a large amount of BChla was solubilized in the acetone (Figure 8a). To accurately quantify the pigments (carotenoids and BChla) released during lysis, fresh cell biomass (2.7 g) was lysed with acetone (1:1), and the dissolved pigments were measured relative to the total intracellular pigment content. The acetone-solubilized fraction 30 minutes after lysis consisted of approximately 0.3% carotenoids and approximately 1% BChla (Figure 8b). To reduce scattering effects, the supernatant was centrifuged before measurement (Figure 9a). As a result, absorption peaks were detected around 750 nm and 525 nm. These were compared to the characteristic BChla peaks in the total pigment fraction at 770 nm (Qy) and 580 nm (Qx) (Figure 9b). Bacteriopheophytin (BPhe), a demetallated derivative of BChla, exhibits absorption bands at 750 nm and 525 nm, and was shown to be present in the acetone extract after 30 minutes. To determine whether BPhe arose from BChla degradation during small-scale processing or was naturally occurring BPhe that solubilizes faster than BChla in acetone, the absorption spectra of the supernatants were compared after acetone fracturing of fresh cell biomass (100 g) for 20 minutes or overnight. The results showed that BChla was primarily solubilized after overnight fracturing, while BPhe was primarily extracted within the first 20 minutes (Figure 9c).BPhe is essentially BChla with two hydrogen atoms substituted for the central magnesium ion, and is less lipophilic than BChla. It was thought that BPhe solubilized more quickly due to its different polarity and aqueous environment because the 1:1 acetone suspension retained water from the fresh cell pellets.

[0090] The color change of the acetone cell suspension was also observed and was found to be dependent on the time of disruption (Figure 8c). To observe the loss of pigment from the cell pellet over time and to reduce the relative water content in the acetone cell suspension, approximately 100 g of fresh cell biomass was treated with acetone (1:1.5), and the absorption spectra of the extract were obtained up to 2.5 hours after resuspension. During this test, the acetone cell suspension (upper panel) and the extract (lower panel) showed a color change from purple to green and from gray to blue (Figure 8c). Normalized absorption spectra at 15 and 30 minutes after resuspension showed that the cell lysate remained stable up to 30 minutes (Figure 8d). Peaks characteristic of BChla (360 nm, 580 nm, 770 nm) were observed in the extract after resuspension at 1 hour, 1.5 hours, 2 hours, and 2.5 hours, indicating that it is the main extractable pigment in acetone (Figure 8d). The absence of a distinct absorption peak around 480 nm indicates minimal carotenoid dissolution in acetone, suggesting the need for further optimization of the process pipeline for carotenoid extraction. On the other hand, the selective extraction behavior demonstrates the usefulness of acetone for extracting specific pigments while minimizing co-extraction of carotenoids, and its ability to be used for cell disruption in closed-loop solvent recovery systems, demonstrating its potential to reduce both cost and environmental impact in industrial workflows.

[0091] Statistical Analysis: The statistical significance of the differences between treatments in this embodiment was determined using one-way analysis of variance (ANOVA) and Tukey post-hoc analysis (GraphPad Prism 9, GraphPad Software, Boston, Massachusetts, USA). P values ​​≤0.05, ≤0.01, ≤0.001, and ≤0.0001 are indicated by *, **, ***, and ****, respectively, and were considered statistically significant. Unless otherwise noted, statistical analyses were derived from the mean values ​​of at least three independent cultures or Processed Biomass (PB) derived therefrom.

Claims

1. A method for crushing marine purple photosynthetic bacteria, comprising the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria, wherein the crushing suppresses changes in the amino acid composition or carotenoid composition of the marine purple photosynthetic bacteria.

2. The crushing method according to claim 1, wherein the crushing of the marine purple photosynthetic bacteria is carried out by immersing the marine purple photosynthetic bacteria in an acetone solvent.

3. The crushing method according to claim 1 or 2, wherein the change in the composition of the amino acids is a change in the ratio of the content of each natural amino acid contained in the crushed material, compared to the ratio of the content of each natural amino acid contained in marine purple photosynthetic bacteria.

4. The crushing method according to claim 1 or 2, wherein the change in the amino acid composition is a change in the total amount of amino acids and / or the total amount of free amino acids contained in marine purple photosynthetic bacteria.

5. The crushing method according to claim 1 or 2, wherein the amount of amino acids contained in the crushed material is 85% by mass or more of the total amount of amino acids contained in marine purple photosynthetic bacteria.

6. The crushing method according to claim 1 or 2, wherein the amount of free amino acids contained in the crushed material is 85% by mass or more of the total amount of free amino acids contained in marine purple photosynthetic bacteria.

7. The crushing method according to claim 1 or 2, wherein the change in the composition of the carotenoids is a change in the total amount of carotenoids contained in marine purple photosynthetic bacteria.

8. The crushing method according to claim 1 or 2, wherein the total amount of carotenoids contained in the crushed material is 90% by mass or more of the total amount of carotenoids contained in marine purple photosynthetic bacteria.

9. The crushing method according to claim 1 or 2, wherein the carotenoid is one or more selected from the group consisting of spheroidenone, spheroidene, dimethylspheroidenone, 3,4-dihydroxyspheroidenone, and neurospolene.

10. The crushing method according to claim 1 or 2, further comprising the step of drying the crushed material.

11. The crushing method according to claim 1 or 2, wherein the crushed material is for use as a nutrient agent.

12. The crushing method according to claim 11, wherein the nutrient is for use as fertilizer, animal feed, food, or culture medium.

13. A method for suppressing changes in the amino acid composition or carotenoid composition of marine purple photosynthetic bacteria, comprising the step of crushing marine purple photosynthetic bacteria in an acetone solvent to obtain a crushed product of marine purple photosynthetic bacteria.

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