Lactobacillus plantarum JYLP-376 microbial inoculum and application thereof in preparation of products for preventing delayed muscle soreness

Abstract

The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum JYLP-376 microbial inoculum and application of the lactobacillus plantarum JYLP-376 microbial inoculum to preparation of products for preventing delayed muscle soreness, and the lactobacillus plantarum JYLP-376 microbial inoculum comprises lactobacillus plantarum JYLP-376, lactobacillus plantarum JYLP-376, lactobacillus plantarum JYLP-376, lactobacillus plantarum JYLP-376, lactobacillus plantarum JYLP-376, lactobacillus plantarum The preservation number of the lactobacillus plantarum JYLP-376 is CGMCC (China General Microbiological Culture Collection Center) The lactobacillus plantarum JYLP-376 microbial inoculum can remove excess oxygen free radicals in the body by effectively adjusting calcium ion metabolism in muscle cells and the content of CK, MDA and SOD in serum, enhance the load bearing capacity of the muscle cells for movement, prevent muscle cell damage, maintain the movement function and achieve the effect of preventing delayed muscular soreness to a certain extent.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a lactobacillus plantarum JYLP-376 bacterial agent and application thereof in preparing a product for preventing delayed onset muscle soreness. Background Art

[0002] When the human body bears high-intensity exercise load, muscles will experience soreness accompanied by discomfort symptoms such as stiffness and swelling. The main feature of soreness is that the soreness symptoms do not occur during or immediately after exercise, but begin to occur about 24 hours after exercise, so it is called delayed muscle soreness (DOMS). The soreness of DOMS generally lasts for 1-4 days and then gradually disappears. It is a special type of exercise-induced muscle fatigue. DOMS is most common in high-intensity, long-term centrifugal exercise. Centrifugal exercise causes delayed ultrastructural changes in skeletal muscles such as skeletal muscle sarcolemma, myofibrils, and mitochondrial morphology, increased muscle-related enzyme activity, and increased muscle protein decomposition products in the blood and urine, causing micro-damage to exercise muscles and triggering skeletal muscle soreness.

[0003] Studies have found that DOMS and calcium ions (Ca 2+ ) metabolism. When the human body performs eccentric contraction exercise, the external load increases, and the muscle fibers produce greater contraction force by lengthening. When the muscles contract continuously or excessively, it will cause calcium ion metabolism disorder, and the Ca outside the muscle cell membrane will increase. 2+ Continue to enter the muscle cells, causing the cytoplasmic Ca 2+ Overload. Cytosolic Ca 2+ Overload will activate a variety of enzymes, promote the hydrolysis of various proteins in muscle cells, and cause muscle cell damage. 2+ Overload can also cause mitochondrial Ca 2+ The increased concentration causes mitochondrial swelling, which reduces mitochondrial related functions, resulting in further energy reduction, and ultimately leads to muscle cell damage and decreased motor function. At the same time, when DOMS occurs, the content of inflammatory factors such as interleukin 1 and interleukin 6 increases, causing pain receptors to activate, resulting in persistent inflammatory reactions such as redness, swelling, heat, and pain in local muscles.

[0004] Currently, there are many intervention measures for DOMS, including anti-inflammatory drugs, ultrasound, electrical stimulation, hyperbaric oxygen, pressure bandages, massage and exercise therapy, which can relieve DOMS after high-intensity exercise, but their preventive effects on DOMS before high-intensity exercise are not satisfactory. Summary of the invention

[0005] In view of the technical problem that the prior art has a poor effect on preventing DOMS, the present invention provides a Lactobacillus plantarum JYLP-376 bacterial agent and its use in preparing a product for preventing delayed onset muscle soreness. The Lactobacillus plantarum JYLP-376 bacterial agent can effectively prevent delayed onset muscle soreness by regulating calcium ion metabolism in skeletal muscle cells.

[0006] In a first aspect, the present invention provides a use of a Lactobacillus plantarum JYLP-376 bacterial agent in the preparation of a product for preventing delayed onset muscle soreness, wherein the Lactobacillus plantarum JYLP-376 bacterial agent comprises Lactobacillus plantarum ( Lactobacillus plantarum ) JYLP-376; Lactobacillus plantarum JYLP-376 was deposited in the General Microbiology Center of China Culture Collection Administration on July 8, 2019. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.18099.

[0007] Products for preventing delayed onset muscle soreness are medicines for preventing delayed onset muscle soreness.

[0008] Furthermore, delayed onset muscle soreness includes muscle soreness caused by skeletal muscle damage or necrosis, which occurs 24-36 hours after eccentric exercise, due to cytoplasmic calcium overload, mitochondrial swelling, and / or increased creatine kinase in serum.

[0009] Furthermore, the prevention of delayed onset muscle soreness includes regulating the levels of creatine kinase, malondialdehyde and superoxide dismutase in serum.

[0010] Furthermore, prevention of delayed onset muscle soreness involves intervention of the inflammatory response of skeletal muscle.

[0011] Furthermore, intervention in the inflammatory response of skeletal muscle is to accelerate the release of IL-6.

[0012] Furthermore, preventing delayed onset muscle soreness involves reducing calcium levels within muscle cells.

[0013] Furthermore, reducing the calcium ion content in muscle cells means reducing the cytoplasmic calcium ion content of skeletal muscle cells.

[0014] Furthermore, reducing the calcium ion content in muscle cells means reducing the calcium ion content in mitochondria of skeletal muscle cells.

[0015] In a second aspect, the present invention provides a Lactobacillus plantarum JYLP-376 bacterial agent. The Lactobacillus plantarum JYLP-376 is inoculated in an MRS liquid culture medium, the bacterial bodies are collected by centrifugation, washed, and then resuspended in reconstituted skim milk, and then freeze-dried and mixed with maltodextrin to obtain the Lactobacillus plantarum JYLP-376 bacterial agent for preventing delayed onset muscle soreness.

[0016] Furthermore, the number of bacteria in the Lactobacillus plantarum JYLP-376 agent was 5×10 9 cfu / g.

[0017] The beneficial effects of the present invention are: The plant lactobacillus JYLP-376 bacterial agent provided by the present invention can remove excess oxygen free radicals in the body by effectively regulating calcium ion metabolism in muscle cells and the contents of CK, MDA and SOD in serum, enhance the load bearing capacity of muscle cells to exercise, prevent muscle cell damage, maintain motor function, and play the effect of preventing delayed muscle soreness to a certain extent. For damaged muscle cells, it can also accelerate the early release of inflammatory factor IL-6 after exercise, and play the role of preventing delayed muscle soreness in a way of accelerating the inflammatory process. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0019] Example 1 Lactobacillus plantarum JYLP-376 bacterial agent (1) Preparation of MRS liquid medium and MRS plate medium MRS liquid culture medium includes 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of Tween 80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, and 1000mL of distilled water; after mixing the above raw materials, adjusting the pH to 6.8, stirring evenly, and sterilizing at 121°C and 0.1MPa for 20min to obtain MRS liquid culture medium; The MRS plate culture medium includes 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween 80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 15 g of agar, and 1000 mL of distilled water. The above raw materials are mixed, the pH is adjusted naturally, the mixture is stirred evenly, and the mixture is sterilized at 121° C. and 0.1 MPa for 20 min, poured into a plate, and cooled to obtain the MRS plate culture medium.

[0020] (2) Preparation of Lactobacillus plantarum JYLP-376 powder The preserved Lactobacillus plantarum JYLP-376 was activated on an MRS plate medium, and the activated Lactobacillus plantarum JYLP-376 was inoculated into an MRS liquid medium at a rate of 1.0 wt %. After culturing at 37 ° C for 24 h, the cells were collected by centrifugation, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk (manufacturer: Henan Huaxing Biotechnology Co., Ltd.) to obtain a suspension; the concentration of the suspension was adjusted to 1.0 × 10 10 cfu / mL, a bacterial suspension was obtained, and the bacterial suspension was freeze-dried to obtain Lactobacillus plantarum JYLP-376 powder.

[0021] (3) Preparation of Lactobacillus plantarum JYLP-376 bacterial agent The Lactobacillus plantarum JYLP-376 bacterial powder was mixed with maltodextrin (manufacturer: Baolingbao Biotechnology Co., Ltd.) to prepare the Lactobacillus plantarum JYLP-376 bacterial agent.

[0022] The number of Lactobacillus plantarum JYLP-376 bacteria was detected by plate counting method and was 5×10 9 cfu / g.

[0023] Comparative Example 1 Lactobacillus plantarum ATCC8014 bacterial agent Lactobacillus plantarum ATCC8014 was activated on an MRS plate medium (the formulation and preparation method are shown in Example 1), and the activated Lactobacillus plantarum ATCC8014 was inoculated into an MRS liquid medium at a rate of 1.0 wt %, and cultured at 37° C. for 24 h, and then the cells were collected by centrifugation, washed with sterile saline, and resuspended in 15% (w / w) reconstituted skim milk (manufacturer: Henan Huaxing Biotechnology Co., Ltd.) to obtain a suspension; the concentration of the suspension was adjusted to 1.0×10 10 cfu / mL, a bacterial suspension was obtained, and the bacterial suspension was freeze-dried and mixed with maltodextrin (manufacturer: Baolingbao Biotechnology Co., Ltd.) to prepare a Lactobacillus plantarum ATCC8014 bacterial agent.

[0024] The number of Lactobacillus plantarum ATCC8014 bacteria was detected by plate count method and was 5×10 9 cfu / g.

[0025] Experimental Example 1 Experiment 1 on the prevention of delayed onset muscle soreness by using Lactobacillus plantarum JYLP-376 bacterial agent During high-intensity centrifugal exercise, the body is ischemic, hypoxic, and energy consumption increases, resulting in insufficient ATP supply, which in turn causes enzyme inactivation, decreased ATP-dependent ion pump function, and increased cytoplasmic Ca 2+ Ion abnormalities, intramitochondrial Ca 2+Accumulation inhibits mitochondrial respiration, increases anaerobic energy consumption, and ultimately leads to muscle cell damage and even necrosis, resulting in delayed muscle soreness.

[0026] 1. Construction of rat model for prevention of delayed onset muscle soreness 1.1 Grouping 80 6-week-old male SPE rats weighing 120±2g were purchased from Changsha Tianqin Biotechnology Co., Ltd. and were subjected to adaptive feeding for 2 weeks. The feeding conditions were: sufficient food and water, temperature of 25°C, 12h bright, 12h dark, and no acute or endurance training was performed on the rats during this period. After 2 weeks of adaptation, the rats were randomly divided into 4 groups for administration, with 20 rats in each group, including exercise group, non-exercise group, Example 1 group, and Comparative Example 1 group.

[0027] 1.2 Administration Example 1 Rats in group 1 were gavaged with 4×10 7 cfu / mL of Lactobacillus plantarum JYLP-376 agent; the rats in the comparative example 1 group were gavaged with the Lactobacillus plantarum ATCC8014 agent prepared in comparative example 1 dissolved in normal saline (the gavage amount was the same as that of the rats in the example 1 group); the rats in the exercise group and the non-exercise group were gavaged with the same volume of normal saline, and the feeding was continued for 21 days.

[0028] 1.3 Establishment of a rat model of exhaustive eccentric exercise injury The rats in the exercise group, Example 1 group and Comparative Example 1 group were subjected to acute downhill running exercise, with a slope of -16° and a speed of 16 m / min, and continued to exercise for 90 min, while the rats in the non-exercise group only performed normal autonomous activities. After the acute downhill running exercise, 10 rats were randomly selected from each group and placed in corresponding mouse cages for conventional feeding, and the remaining 10 rats in each group were anesthetized with sodium pentobarbital (anesthetic dosage was 3 mg / 100 g body weight) at 36 h after the acute downhill running exercise, and various indicators were measured.

[0029] 1.4 Index determination 1.4.1 Determination of serum creatine kinase (CK), malondialdehyde (MDA), and superoxide dismutase (SOD) levels Most of the CK in serum comes from muscles that are closely related to exercise. Changes in its activity can reflect the response of muscle cells to exercise load, and then reflect the degree of muscle cell damage.

[0030] After anesthesia, 2 mL of venous blood was drawn from each rat, and the serum was separated after standing for 30 minutes. The levels of CK, MDA and SOD in the serum were determined within 3 hours.

[0031] 1.4.2 Determination of free calcium content in mitochondria Expose the hind limb muscles of the rat under an ice bath (ice tray), take the left quadriceps red muscle (mainly the deep vastus intermedius and vastus medialis, one of the skeletal muscles), quickly remove excess fat and connective tissue, weigh, wrap it with cut and marked tin foil, put it into liquid nitrogen (30 minutes), and then grind it into muscle powder in liquid nitrogen. Then transfer the muscle powder to a -80℃ ultra-low temperature refrigerator for storage and standby.

[0032] Preparation of mitochondrial suspension: 1) Take 300 mg of muscle powder, add 3 mL of pre-cooled (4°C) solution I (0.12 M KCl, 20 mM Hepes, 5 mM MgCl2, 1 mM EDTA, pH 7.4), mix well to prepare tissue homogenate, and centrifuge at 4°C and 600 g for 10 min; 2) Take the supernatant and filter it through double-layer gauze to remove fat. Store the filtrate at 4°C. Add 2 mL of solution I to the precipitate after centrifugation and resuspend it. 3) Centrifuge at 4°C, 600 g for 10 min, take the supernatant and filter it with double-layer gauze, mix the filtrate with the filtrate in 2), and centrifuge at 4°C, 17,000 g for 10 min; 4) Discard the supernatant, keep the pellet, add 5 mL of solution I (add 250 μL 0.1 g / mL defatted BSA) to resuspend, centrifuge at 4°C and 7500 g for 10 min, discard the supernatant, keep the pellet, add 300 μL of solution II (0.3 M sucrose, 2.0 mM Hepes, 0.1 mM EDTA, 2 mg defatted BSA / mL, pH 7.4), and carefully pipette until suspended. This is the mitochondrial suspension.

[0033] Mitochondrial free Ca 2+ Concentration determination: Take the mitochondrial suspension and mix it with Fura-2 AM working solution in a ratio of 1:1, incubate it at 37℃ in the dark for 45min, add 4mL solution II, centrifuge at 12000g for 2min, discard the supernatant, keep the precipitate, and resuspend it with solution II. Repeat twice, and use a fluorescence spectrometer to detect the fluorescence intensity of the resuspension. The excitation wavelengths are 340nm and 380nm, respectively, and the emission wavelength is 510nm. The fluorescence intensity F340 and F380 of the resuspension are measured respectively, and the measured images are processed and quantified using Origin8.0 image processing software.

[0034] 1.4.3 Determination of cytoplasmic calcium content in skeletal muscle cells Take the quadriceps femoris of rats (one of the skeletal muscles), rinse with double distilled water, cut into several pieces, and put them into an electric hot air drying oven (manufacturer: Shanghai Yiheng Scientific Instrument Co., Ltd.) to dry to constant weight, and then measure the weight of the sample. Put the weighed samples into beakers, add 10mL of concentrated nitric acid (super pure), heat and nitrate, use a 100mL volumetric flask to make up the volume, and finally use HiTAChi polarized Zeeman atomic absorption spectrophotometer (manufacturer: Hitachi, Japan) to determine the Ca content in the samples. 2＋ concentration.

[0035] Skeletal muscle Ca 2＋ The concentration calculation formula is as follows:

[0036] 2.1 Results of determination of CK, MDA and SOD levels in serum As can be seen from Table 1, compared with the non-exercise group, the CK content in the serum of the exercise group rats increased significantly (p<0.05), indicating that after centrifugal exercise, skeletal muscle cells were locally damaged, which increased the overflow of muscle enzymes and led to an increase in the CK content in the serum. Compared with the exercise group, the CK content of Example 1 group was significantly reduced, indicating that rats were continuously gavaged with plant lactobacillus JYLP-376 for three weeks before exercise, which could enhance the load bearing capacity of skeletal muscle cells for exercise, and to a certain extent prevent delayed muscle soreness. However, the CK content of the comparative example 1 group did not decrease significantly compared with the exercise group, indicating that the plant lactobacillus ATCC8014 bacterial agent could not regulate the increase in CK content and skeletal muscle cell damage caused by muscle enzyme overflow, that is, rats were continuously gavaged with plant lactobacillus ATCC8014 bacterial agent before exercise, which could not prevent delayed muscle soreness.

[0037] Similarly, compared with the non-exercise group, the MDA content in the rats in the exercise group increased, and the SOD content decreased, and it was significant (p<0.05), indicating that the oxygen free radicals in the body increased after high-intensity centrifugal exercise, resulting in delayed muscle soreness after exercise. Compared with the exercise group, the MDA content in the serum of the rats in Example 1 group decreased, and the SOD content increased, indicating that the rats were continuously gavaged with plant lactobacillus JYLP-376 for three weeks before exercise, which could remove excess oxygen free radicals in the body and reduce the damage of high-intensity centrifugal exercise to the body, and to a certain extent, it played a role in preventing delayed muscle soreness. However, the MDA and SOD contents of the comparative example 1 group did not change significantly compared with the exercise group, indicating that the continuous gavage of plant lactobacillus ATCC8014 inoculum in rats before exercise could not prevent delayed muscle soreness.

[0038] Table 1 Contents of CK, MDA and SOD in serum (n=10, mean ± SD)

[0039] Note: * Compared with the non-exercise control group, p < 0.05; # Compared with the exercise control group, p<0.05.

[0040] 2.2 Results of free calcium content in skeletal muscle and mitochondria As shown in Table 2, the Ca2+ levels in the cytoplasm and mitochondria of the rats in the exercise group after an acute downhill run were 2+ The contents of the two groups were significantly higher than those of the non-exercise group, indicating that after high-intensity centrifugal exercise, the cytoplasmic Ca 2+ Rapid increase in mitochondrial energy metabolism inhibits mitochondrial Ca 2+ The buffering capacity of cytosolic Ca 2+ As the concentration increases, the Ca 2+ The concentration will also increase, inhibiting mitochondrial related functions, leading to muscle cell damage, fatigue, pain, and decreased motor function.

[0041] Example 1: Ca in the cytoplasm and mitochondria of skeletal muscle of rats in group 2+ The content was significantly lower than that in the exercise group (p<0.05). This indicates that continuous intragastric administration of Lactobacillus plantarum JYLP-376 for three weeks before exercise can significantly reduce the Ca2+ content in the cytoplasm and mitochondria of rats after high-intensity centrifugal exercise. 2+ The increase in the content of Ca2+ in the cytoplasm and mitochondria of the skeletal muscle cells in the control group 1 was significantly lower than that in the control group 2. 2+ The content of Lactobacillus plantarum ATCC8014 inoculum did not change significantly compared with the exercise group, indicating that continuous intragastric administration of Lactobacillus plantarum ATCC8014 before exercise could not regulate Ca 2+ Metabolism within skeletal muscle cells plays a role in preventing delayed onset muscle soreness.

[0042] Table 2 Calcium ion levels in cytoplasm and mitochondria of skeletal muscle cells after 36h of exercise (n=10, mean±SD)

[0043] Note: * Compared with the non-exercise group, p<0.05, # Compared with the exercise group, p<0.05.

[0044] Experimental Example 2 Experiment 2 on the prevention of delayed onset muscle soreness by using Lactobacillus plantarum JYLP-376 1. Determination of inflammatory factor IL-6 content The inflammatory factor IL-6 content was measured in the rats raised conventionally in each cage in Experimental Example 1. Specifically, 1 mL of abdominal aortic blood was collected from each rat on the 1st, 2nd, 5th and 7th day after exercise, and the plasma was collected after centrifugation to measure the inflammatory factor IL-6 content according to the method in the instructions of the IL-6 test kit (manufacturer: Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.).

[0045] The data were statistically analyzed using SPSS15.0 software.

[0046] 2. Results of determination of inflammatory factor IL-6 content As shown in Table 3, compared with the non-exercise group, the IL-6 content of the exercise group rats on the 1st, 2nd, 5th and 7th day after exercise was significantly increased (p<0.05), indicating that high-intensity eccentric exercise causes inflammation in muscle cells, and the inflammatory factor IL-6 is released into the blood. Compared with the exercise group, the IL-6 content of the rats in Example 1 group on the 1st, 2nd and 5th day after exercise was significantly increased (p<0.05), and the IL-6 content on the 7th day after exercise was significantly reduced (p<0.05), indicating that gavage of plant lactobacillus JYLP-376 before exercise can relieve exercise-induced muscle soreness by accelerating the inflammatory process, shortening the inflammatory response period after injury, and accelerating muscle regeneration. The IL-6 content of the comparative example 1 group did not change significantly compared with the exercise group, indicating that continuous gavage of plant lactobacillus ATCC8014 bacterial agent in rats before exercise cannot prevent delayed muscle soreness by accelerating the inflammatory process.

[0047] Table 3 Concentration of inflammatory factor IL-6 in plasma (n=10, mean±SD)

[0048] Note: * Compared with the non-exercise group, p<0.05, # Compared with the exercise group, p<0.05.

[0049] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily conceive of changes or substitutions within the technical scope disclosed by the present invention, and such changes or substitutions shall be within the scope of protection of the present invention.

Claims

1. An application of a Lactobacillus plantarum JYLP-376 bacterial agent in the preparation of a product for preventing delayed onset muscle soreness, characterized in that: Lactobacillus plantarum JYLP-376 bacterial agent includes Lactobacillus plantarum ( Lactobacillus plantarum ) JYLP-376; the preservation number of Lactobacillus plantarum JYLP-376 is CGMCC NO.18099.

2. The use according to claim 1, characterized in that Delayed onset muscle soreness includes muscle soreness caused by skeletal muscle damage or necrosis, which occurs 24-36 hours after eccentric exercise, due to cytoplasmic calcium overload, mitochondrial swelling, and / or increased serum creatine kinase.

3. The use according to claim 1, characterized in that Prevention of delayed onset muscle soreness involves regulating serum levels of creatine kinase, malondialdehyde, and superoxide dismutase.

4. The use according to claim 1, characterized in that Prevention of delayed onset muscle soreness involves intervention in the inflammatory response of skeletal muscle.

5. The use according to claim 4, characterized in that Intervention of skeletal muscle inflammatory response is to accelerate the release of IL-6.

6. The use according to claim 1, characterized in that Preventing delayed onset muscle soreness involves reducing the amount of calcium ions within muscle cells.

7. The use according to claim 6, characterized in that Reducing the calcium ion content in muscle cells means reducing the cytoplasmic calcium ion content of skeletal muscle cells.

8. The use according to claim 6, characterized in that Reducing the calcium ion content in muscle cells is to reduce the calcium ion content in the mitochondria of skeletal muscle cells.

9. A Lactobacillus plantarum JYLP-376 bacterial agent, characterized in that: Lactobacillus plantarum JYLP-376 was inoculated into MRS liquid culture medium, the bacteria were collected by centrifugation, washed, and resuspended in reconstituted skim milk. After freeze-drying, the bacteria were mixed with maltodextrin to obtain the Lactobacillus plantarum JYLP-376 bacterial agent for preventing delayed onset muscle soreness.

10. The Lactobacillus plantarum JYLP-376 bacterial agent according to claim 9, characterized in that: The number of Lactobacillus plantarum JYLP-376 bacteria is 5×10 9 cfu / g.

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