WO2023135607A1 - A method for preparation of functional curd with anti-ageing property and high shelf life - Google Patents
A method for preparation of functional curd with anti-ageing property and high shelf life Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/121—Brevis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/24—Lactobacillus brevis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
Definitions
- the present invention in general, relates to a method for production of functional curd with anti-ageing property, consistent curdling and high shelf life.
- the present invention relates to a method for production of functional curd applying a specially integrated starter culture which ensures antiageing property, consistent curdling and high shelf life in the end product.
- yoghurt is consumed as a source of probiotics.
- the microbial strains in yoghurts besides showing probiotic potential, additionally show a variety of other health beneficial effects.
- Lactobacillus plantarum JBC5 showed probiotic potential as per ICMR guidelines, and further showed anti-ageing properties based on our experiment on model organism Caenorhabditis elegans.
- the anti-ageing properties in probiotic microbes are well documented, such as the Lactobacillus pentosus var. plantarum and Lactobacillus curvatus C3 KCCM430009 reported to prevent or treat aging.
- Lactobacillus fermentum acts as therapeutic agent to treat respiratory and gastrointestinal diseases. Additionally, Lactobacillus fermentum strain LP3 and LP4 are reported to be a promising starter culture for production of different dairy products.
- the present invention for the first time provides a method for production of functional curd with anti-ageing property, consistent curdling and high shelf life.
- the bacterial strain L. plantarum JBC5 isolated by the inventors possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
- the invention optimizes the cowmilk curd formation by JBC5 in consortium with another bacterium as stated aforesaid.
- cowmilk curd with defined proportions of starter inoculum including Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 having anti-ageing and curdling property. It is another object of the present invention to provide bacterial strain L. plantarum JBC5 possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
- Lactobacillus plantarum JBC5 is isolated from a local variety of yoghurt.
- Lactobacillus brevis H4 is isolated from honey.
- the starter culture is optimized using different colony forming units and selecting the one which stabilized Lactobacillus plantarum JBC5 and increased the shelf life, anti-ageing property and consistent curdling of functional curd.
- the present invention also provides a functional curd obtained by the method as described hereinbefore.
- the bacterial strain L. plantarum JBC5 possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
- Figures la and lb illustrate neighbour-joining tree based on 16S rRNA gene sequences (1297 bases) of (a) L. plantarum strain JBC5, and (b) L. brevis H4 respectively.
- Figure 2a illustrates schematic diagram depicting preparation of curd while Figure 2b illustrates curdling for successive days in control, milk inoculated with JBC5 along with H4.
- Figure 3 in its entirety illustrate effect of L. plantarum JBC5 on longevity and aging biomarkers of C. elegans.
- Figure 4 illustrates results of experiment performed on a 60 mm Petri-plate containing NGM medium for the food preference assay.
- Figure 5a graphically illustrate qRT-PCR analysis on the mRNA expression of genes involved in longevity, anti-oxidation, fat accumulation, and learning and memory
- Figure 5b graphically illustrates mRNA expression of genes involved in innate immunity and tight junction proteins (ZOO-1) in maintaining intestinal integrity against infection with pathogen S. aureus.
- Figure 6 illustrate predicted mechanisms of JBC5 induced anti-aging mechanisms in C. elegans.
- the microbes were isolated from ethnic foods of Northeast India.
- two microbial strain are used in this preparation and among them L. plantarum (Lactobacillus plantarum) JBC5 was isolated from a traditional curd being prepared by Nepali community in Jagi road, Assam and L. brevis (Lactobacillus brevis) H4 was isolated from honey collected from tribal honey hunters of Northeast India.
- plantarum species-specific PCR confirmed its species-specific sequence, potential probiotic marker genes ⁇ encode bile salt hydrolase (bsh) and collagen-binding protein (cbp) and the antimicrobial gene plantaricin-biosynthetic gene (Pin), with amplicon lengths 152, 975, 2174 and 231 bp. All these sequences have been submitted in the NCBI database (MW846636- MW846639).
- Figures la and lb illustrate neighbour-joining tree based on 16S rRNA gene sequences (1297 bases) of (a) L. plantarum strain JBC5, and (b) L. brevis H4 respectively
- L. plantarum JBC5 L. plantarum JBC5
- L. brevis H4 L. brevis H4
- Lactobacillus strains are reported to tolerate pH at 4.6, which is common final acidity of fermented dairy products.
- JBC5 and H4 retained their viability even at pH 1 and pH3. Additionally, their survival percentage was found to be 93.36% and 94.52% in pH2 with pepsin while 99% and 99.32% in pancreatin (pH8).
- JBC5 and H4 tolerated 1% of oxgall for 240 min and also showed its strong resistance to bile salts (Table 1).
- Adhesion assays of L. plantarum JBC5 and L. brevis H4 were performed according to standard protocol as described by Margakoudis et al. 2006. The adhesion of L.
- Antibiotics sensitivity The strains are sensitive against all antibiotics as detailed in ICMR guidelines
- Fresh cow milk was collected from the dairy farm aseptically in a sterilized bag and was bought to laboratory in refrigerated condition (4°C). In laboratory, the milk samples were pasteurized at 100°C for 2 mins ⁇ high temperature short time (HTST) ⁇ . Post-pasteurization the milk was allowed to cool for 30-35 minutes.
- milk and the starter culture were optimized using different colony forming units, which involves the inventive steps of this patent.
- the cow milk is maintained at a moisture content of 89.73% and PH of 6.77 ⁇ 0.057.
- Figure 2a illustrates schematic diagram depicting preparation of curd while Figure 2b illustrates curdling for successive days in control, milk inoculated with JBC5 along with H4.
- Lactobacillus plantarum and Lactobacillus brevis- It is generally regarded as safe according to Food Safety and Standard (FSS) ACT. 2016 schedule VII.
- Anti-ageing activity of L. plantarum JBC5 Probiotic microbes play an important role in maintaining the human health. They perform their beneficial functions on the host's health through different mode of actions, such as competing with pathogens, enhancing gut mucosal immunity, releasing antimicrobial substances and immunomodulation.
- C. elegans is considered as a bacterivore, i.e. feeds on bacterium, therefore the potential of any bacterium on the physiology of C. elegans can be studied.
- the bacterium Lactobacillus plantarum strain JBC5 fed to C. elegans enhanced their mean lifespan by 27.81% compared to control (fed with Escherichia coli OP50) (Figure. 3A) (***p ⁇ 0.0001, log-rank test).
- strain JBC5 also promoted the youngness parameters of C. elegans, including pharynx pumping and body bends.
- the results showed increase in pharyngeal pumping rates and body bends by 179.47 % and 148.66% compared to control-fed in aged day-14 C. elegans (***p ⁇ 0.001 for both pharynx pumps and body bends) ( Figure. 3 B and C).
- the feeding of strain JBC5 also reduced the accumulation of aging pigment lipofuscin and fat storage by 51.79% and 35.77% in aged day-14 C. elegans (***p ⁇ 0.001 for lipofuscin and **p ⁇ 0.01 for fat accumulation) (Figure. 3 D-G).
- Figure.3 illustrate effect of L. plantarum JBC5 on longevity and aging biomarkers of C. elegans.
- C. elegans were pre-cultured on JBC5 or control for 3 days and transferred to nematode growth media (NGM) plates seeded with pathogen S. aureus and their survival was analyzed.
- NNM nematode growth media
- the pre-culturing of C. elegans on strain JBC5 increased their survival against pathogen S. aureus by 25% compared control-fed (**p ⁇ 0.01) ( Figure. 4D).
- FIG. 4 A Schematic diagram showing the experimental design. The experiment was performed on a 60 mm Petri-plate containing NGM medium for the food preference assay. For food preference assay, 3-days old C. elegans were transferred onto the center of the plates equidistant from both the bacterial lawns and counted after 4 hours of incubation at 20 °C. For training, the C. elegans were transferred to NGM plates seeded with LPJBC5 for 4 hours and kept at 20 °C. B and C.
- the binary food choice assay was performed as Choice index (CI) was calculated for C. elegans.
- Memory index (CI trained- CI naive) was calculated from the results of the binary choice assay (Scale bar, 1 mm). Error bars represent mean ⁇ SEM.
- CI -1.0 shows complete food preference for control food OP50.
- CI + 1.0 shows complete food preference for testing bacteria JBC5.
- CI 0.0 shows equal distribution of food preference for both OP50 or JBC5.
- strain JBC5-induced longevity mechanisms in C. elegans was studied. Previous studies suggested that mainly two molecular pathways operate in order to extend longevity, i.e. p38 MAPK and DAF-2/DAF-16 pathways (Kumar et al. 2020). Our studies on the mutants of C. elegans suggested that strain JBC5 were able to extend longevity in loss-of-function daf-2 (short-lived) and daf-16 (long- lived) mutants (daf-2, ***p ⁇ 0.0001; daf-16, ***p ⁇ 0.0001 by the log-rank test) (Data not shown). We observed no extension in the longevity of C.
- transcription factor SKN-1 upregulates the expression of anti-oxidative genes, namely catalases (ctl-1 and ctl-2), trx-1, GSTs (gst-4 and gst-7) and SODs (sod-1 and sod-3) ( Figure. 5a).
- FIG. 5b illustrates qRT-PCR analysis on the mRNA expression of genes involved in longevity, anti-oxidation, fat accumulation, and learning and memory.
- Figure 5b illustrates. mRNA expression of genes involved in innate immunity and tight junction proteins (ZOO-1) in maintaining intestinal integrity against infection with pathogen S. aureus. mRNA expression was normalized using the house-keeping gene act-1. Error bars represent ⁇ SEM and each group were statistically analyzed using Student's t-test (*p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001).
- strain JBC5 activates p38 MAPK signaling cascade in C. elegans which further activates its downstream gene skn-1 involved in phase-II detoxification, anti-pathogenic genes and intestinal integrity. Feeding of strain JBC5 reduced the expression of fat accumulation genes and increased the expression of serotonin signaling genes involved in learning and memory ( Figure 6).
- strain JBC5 activates p38 MAPK signaling cascade which further activates the downstream SKN-1 transcription factor involved in phase-II detoxification or antioxidation. These higher expressions of antioxidative genes promoted longevity, increased the expression of genes involved in stress resistance and infection against pathogen S. aureus.
- the feeding of strain JBC5 also reduces the expression of fat-related genes as well as improve the expression of serotonin-signaling genes involved in learning and memory. In result, the feeding of strain JBC5 extends longevity as well as promote reduces other age-associated pathologies in C. elegans.
- the present invention provides: a) method for production of functional curd with anti-ageing property, consistent curdling and high shelf life. b) preparing cowmilk curd with defined proportions of starter inoculum including Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 having anti-ageing and curdling property. c) bacterial strain L. plantarum JBC5 possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
Abstract
The present invention provides a method for preparation of functional curd with anti-ageing property and high shelf life. It comprises collecting fresh cow milk from a dairy farm aseptically in a sterilized bag and maintaining refrigerated condition (4°C). Thereafter pasteurizing is done at 100°C for 2 minutes and the milk is allowed to cool. According to the invention, the said milk is fermented with a starter culture comprising Lactobacillus plantarum JBC5 and Lactobacillus brevis H4. The ratio of Lactobacillus plantarum JBC5: Lactobacillus. brevis H4 = 109cfu/ml in 1:1 concentration. Finally, the desired functional curd is obtained.
Description
A METHOD FOR PREPARATION OF FUNCTIONAL CURD WITH ANTI-AGEING PROPERTY AND HIGH SHELF LIFE
This application claims priority from Indian patent application no. 202231001501 dated 11th January 2022 and the disclosure therein is referred to here in its entirety.
FIELD OF THE INVENTION
The present invention in general, relates to a method for production of functional curd with anti-ageing property, consistent curdling and high shelf life.
In particular, the present invention relates to a method for production of functional curd applying a specially integrated starter culture which ensures antiageing property, consistent curdling and high shelf life in the end product.
BACKGROUND OF THE INVENTION
Globally, yoghurt is consumed as a source of probiotics. The microbial strains in yoghurts besides showing probiotic potential, additionally show a variety of other health beneficial effects.
In Northeast India, majority of ethnic populations prefer consuming curd prepared in artisanal way. Rearing of farm animals such as cow, goat, buffalo etc. is an ethnic and religious practice among the Nepali communities residing in Nepal and also in North-eastern region of India and the biggest supply of cow milk in Northeast India is provided by the Nepalese people, who have one of the major settlement in Jagi road of Assam. The community in Jagi road, besides supplying cow milk, makes a variety of other dairy products and among them Dahi is very popular.
Traditionally, it is believed that their Dahi keeps human healthy, increases stamina, and also increases the longevity of people. The bacterium, Lactobacillus plantarum JBC5 showed probiotic potential as per ICMR guidelines, and further showed anti-ageing properties based on our experiment on model organism
Caenorhabditis elegans. The anti-ageing properties in probiotic microbes are well documented, such as the Lactobacillus pentosus var. plantarum and Lactobacillus curvatus C3 KCCM430009 reported to prevent or treat aging.
Similarly, Lactobacillus fermentum acts as therapeutic agent to treat respiratory and gastrointestinal diseases. Additionally, Lactobacillus fermentum strain LP3 and LP4 are reported to be a promising starter culture for production of different dairy products.
However, in prior art known in this field there is no cowmilk curd with anti ageing, consistent curdling and high shelf life properties all together which is a coveted requirement for long
The present invention for the first time provides a method for production of functional curd with anti-ageing property, consistent curdling and high shelf life.
This is done by applying a fermenting starter culture comprising Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 in a specific concentration, whereby the end product has anti-ageing property, consistent curdling and high shelf life.
The bacterial strain L. plantarum JBC5 isolated by the inventors possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique. The invention optimizes the cowmilk curd formation by JBC5 in consortium with another bacterium as stated aforesaid.
OBJECTS OF THE INVENTION
It is the principal object of the present invention to provide a method for production of functional curd with anti-ageing property, consistent curdling and high shelf life.
It is another important object of the present invention to prepare cowmilk curd with defined proportions of starter inoculum including Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 having anti-ageing and curdling property.
It is another object of the present invention to provide bacterial strain L. plantarum JBC5 possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
How the foregoing objects are achieved and the other aspects of the present invention, will be clear from the following description which is purely by way of understanding and not by way of any sort of limitation.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method for preparation of functional curd with anti-ageing property and high shelf life comprising : a) collecting fresh cow milk from a dairy farm aseptically in a sterilized bag, b) maintaining refrigerated condition (4°C), c) pasteurizing at 100°C for 2 minutes and allowing it to cool, d) fermenting the said milk with a starter culture comprising Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 such that the ratio of Lactobacillus plantarum JBC5: Lactobacillus, brevis H4 = 109cfu/ml in 1 : 1 concentration and obtaining the desired functional curd.
Preferably, Lactobacillus plantarum JBC5 is isolated from a local variety of yoghurt.
More preferably, Lactobacillus brevis H4 is isolated from honey.
Even more preferably, the starter culture is optimized using different colony forming units and selecting the one which stabilized Lactobacillus plantarum JBC5 and increased the shelf life, anti-ageing property and consistent curdling of functional curd.
The present invention also provides a functional curd obtained by the method as described hereinbefore.
The present invention also provides a starter inoculum for preparing cow milk curd having anti-ageing, high shelf and excellent curdling properties comprising Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 having anti-ageing and curdling property wherein the ratio of Lactobacillus plantarum JBC5: Lactobacillus, brevis H4 = 109cfu/ml in 1 : 1.
Preferably, the bacterial strain L. plantarum JBC5 possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The nature and scope of the present invention, will be better understood from the accompanying drawings, which are by way of illustration of some preferred embodiments and not by way of any sort of limitation. In the accompanying drawings:-
Figures la and lb illustrate neighbour-joining tree based on 16S rRNA gene sequences (1297 bases) of (a) L. plantarum strain JBC5, and (b) L. brevis H4 respectively.
Figure 2a illustrates schematic diagram depicting preparation of curd while Figure 2b illustrates curdling for successive days in control, milk inoculated with JBC5 along with H4.
Figure 3 in its entirety illustrate effect of L. plantarum JBC5 on longevity and aging biomarkers of C. elegans.
Figure 4 illustrates results of experiment performed on a 60 mm Petri-plate containing NGM medium for the food preference assay.
Figure 5a graphically illustrate qRT-PCR analysis on the mRNA expression of genes involved in longevity, anti-oxidation, fat accumulation, and learning and memory
Figure 5b graphically illustrates mRNA expression of genes involved in innate immunity and tight junction proteins (ZOO-1) in maintaining intestinal integrity against infection with pathogen S. aureus.
Figure 6 illustrate predicted mechanisms of JBC5 induced anti-aging mechanisms in C. elegans.
DETAILED DESCRIPTION OF THE INVENTION
The following describes some preferred embodiments by way of understanding and not by way of any limitation. Furthermore, the results provided here in after are all exemplary and should not be construed as any sort of restriction.
Isolation and identification of the microbial strains
The microbes were isolated from ethnic foods of Northeast India. In particular, two microbial strain are used in this preparation and among them L. plantarum (Lactobacillus plantarum) JBC5 was isolated from a traditional curd being prepared by Nepali community in Jagi road, Assam and L. brevis (Lactobacillus brevis) H4 was isolated from honey collected from tribal honey hunters of Northeast India.
Phenotypic (Gram's stain, catalase test etc.) and genotypic identification based on 16 rRNA gene sequencing followed by similarity search in NCBI database using BLASTN suite confirmed them L. plantarum and L. brevis. The distinct cluster of L. plantarum and L. brevis within their monophyletic species clade confirmed them being distinct strains (Figures la, b). The 16S rDNA sequence of strain JBC5 have been submitted to the NCBI database (GenBank Acc. No. MG824976.1). L. plantarum species-specific PCR confirmed its species-specific sequence, potential probiotic marker genes {encode bile salt hydrolase (bsh) and collagen-binding protein (cbp) and the antimicrobial gene plantaricin-biosynthetic
gene (Pin), with amplicon lengths 152, 975, 2174 and 231 bp. All these sequences have been submitted in the NCBI database (MW846636- MW846639).
For L. brevis H4, the isolate was extracted from honey and identified by phenotypic and genotypic (Figure lb) approach as mentioned above.
Figures la and lb illustrate neighbour-joining tree based on 16S rRNA gene sequences (1297 bases) of (a) L. plantarum strain JBC5, and (b) L. brevis H4 respectively
Assessment of probiotic potential of L. plantarum JBC5 (LPJBC5) and L. brevis H4 (LBH4)
Probiotic assays of the bacteria were performed for simulated gastrointestinal condition and antibiotic sensitivity as per ICMR guidelines. The results of the various assays are as follows:
In-vitro assessment of in-vitro gastro intestinal (GI) stress tolerance
Lactobacillus strains are reported to tolerate pH at 4.6, which is common final acidity of fermented dairy products. However, JBC5 and H4 retained their viability even at pH 1 and pH3. Additionally, their survival percentage was found to be 93.36% and 94.52% in pH2 with pepsin while 99% and 99.32% in pancreatin (pH8). JBC5 and H4 tolerated 1% of oxgall for 240 min and also showed its strong resistance to bile salts (Table 1). Adhesion assays of L. plantarum JBC5 and L. brevis H4 were performed according to standard protocol as described by Margakoudis et al. 2006. The adhesion of L. plantarum JBC5 with intestinal cell line (HT-29) after 4 hours of incubation was 92.62%. Adhesion of L. brevis H4 was found to be 92.68%.
Table 1. Survival and adhesion of L. plantarum JBC5 and L. brevis H4 under in vitro simulated gastro-intestinal conditions.
* Survival percentage (%) is expressed as the percentage of 1- [(log CFU per ml at T= initial) - (log CFU per ml at T = incubation hour) I (log CFU per ml at T = initial)].
Antibiotics sensitivity: The strains are sensitive against all antibiotics as detailed in ICMR guidelines
Process
Preparation of curd
Fresh cow milk was collected from the dairy farm aseptically in a sterilized bag and was bought to laboratory in refrigerated condition (4°C). In laboratory, the milk samples were pasteurized at 100°C for 2 mins {high temperature short time (HTST)}. Post-pasteurization the milk was allowed to cool for 30-35 minutes. For the preparation of probiotic curd, milk and the starter culture were optimized using different colony forming units, which involves the inventive steps of this patent. Preferably, the cow milk is maintained at a moisture content of 89.73% and PH of 6.77 ± 0.057.
It was found that the milk was best fermented by a combined starter culture containing the bacterial strains i_. plantarum JBC5 and L. brevis H4 having initial log CFU 109 of each strains in comparison to only milk fermented with only Lactobacillus plantarum JBC5. Different combinations of milk and starter culture were used to stabilize L. plantarum JBC5 and eventually increase the shelf-life of curd. It was observed that when the milk was inoculated with only L. plantarum
JBC5 as starter culture, the organoleptic properties are inconsistent and the shelf-life of curd was only 3 days with successive reduction of L. plantarum JBC5 colony count (Figure 2 b). It revealed that JBC5 has poor curdling properties and fails to keep the firmness of curd beyond 3 days. To fix this issue, curdling of milk was performed aiding Lactobacillus brevis H4 in different proportions as shown in table2.
Milk fermented with starter culture with 109 log CFU/ml of each isolates, the firmness, texture, and organoleptic properties of curd improved. Most importantly, both JBC5 and H4 showed stable colony count that persisted for 15 days (Table 3). This clearly revealed that curd prepared with co-culturing of/.. plantarum and L. brevis showed consistent curdling in comparison to the curd prepared with only JBC5. Sensory analysis of the curd is depicted in Table 4.
Table 2. Details of the optimized parameters for preparing curd
Table 3. pH and bacterial colony count (in log CFU scale) in control versus curd inoculated with JBC5, and curd inoculated with JBC5 + H4 on successive days after preparation.
Table 4. Organoleptic properties of curd with starter inoculum 3
*In the scale of 1-10 (<1: unacceptable, >1 to <5:acceptable and >5 to 10: no criticism)
The flow chart of the preparation of curd with new starter culture as defined here and the physical appearance of curd in different sampling days compared to control is shown in figures la, b.
Figure 2a illustrates schematic diagram depicting preparation of curd while Figure 2b illustrates curdling for successive days in control, milk inoculated with JBC5 along with H4.
In-vivo studies in animal model (Caenorhabditis elegans) validated it's safety and efficacy
Lactobacillus plantarum and Lactobacillus brevis-. It is generally regarded as safe according to Food Safety and Standard (FSS) ACT. 2016 schedule VII.
Anti-ageing activity of L. plantarum JBC5: Probiotic microbes play an important role in maintaining the human health. They perform their beneficial functions on the host's health through different mode of actions, such as
competing with pathogens, enhancing gut mucosal immunity, releasing antimicrobial substances and immunomodulation.
However, the concept of probiotic-induced longevity in the host is still not much explored and we focused on developing probiotics for healthy ageing. We employed Caenorhabditis elegans as an invertebrate experimental model for testing anti-aging potential of probiotics. C. elegans is considered as a bacterivore, i.e. feeds on bacterium, therefore the potential of any bacterium on the physiology of C. elegans can be studied. The bacterium Lactobacillus plantarum strain JBC5 fed to C. elegans enhanced their mean lifespan by 27.81% compared to control (fed with Escherichia coli OP50) (Figure. 3A) (***p < 0.0001, log-rank test).
Additionally, the feeding of strain JBC5 also promoted the youngness parameters of C. elegans, including pharynx pumping and body bends. The results showed increase in pharyngeal pumping rates and body bends by 179.47 % and 148.66% compared to control-fed in aged day-14 C. elegans (***p < 0.001 for both pharynx pumps and body bends) (Figure. 3 B and C). Furthermore, the feeding of strain JBC5 also reduced the accumulation of aging pigment lipofuscin and fat storage by 51.79% and 35.77% in aged day-14 C. elegans (***p < 0.001 for lipofuscin and **p < 0.01 for fat accumulation) (Figure. 3 D-G).
Figure.3 illustrate effect of L. plantarum JBC5 on longevity and aging biomarkers of C. elegans.
Feeding of strain JBC5 extended the longevity (A), parameters of young ness, including pharynx pumps and body bends (B and C) as well as accumulation of aging pigment lipofuscin (D and E) (Scale bar, 250 pm) and fat storage (F and G) (Scale bar, 100 pm). Error bars represent ± SEM and each group were statistically analyzed using Student's t-test (*p < 0.05, **p < 0.01 and ***p < 0.001).
The binary choice assay was performed to investigate the effect of strain JBC5 on learning and memory. Adult day-3 C. elegans (N = 30) were transferred onto the center of culturing plates and plates were seeded with control strain and strain JBC5. The choice index (CI) of bacterial food preference was calculated as number of C. elegans in strain JBC5 - number of C. elegans in control strain/ Total number of C. elegans used in assay. Initially, the C. elegans showed no preference for L. plantarum JBC5 (Choice index CI= +0.12) (p > 0.05), while the C. elegans showed feeding preference for strain JBC5 if pre-cultured on it for 4 hours (CI = +0.56) (***p < 0.001) (Figure. 4 A and B).
Further the memory index of trained C. elegans on strain JBC5 over control bacterium were calculated as choice index (C. elegans trained on JBC5) - choice index (C. elegans only grown on control bacterium). The results showed higher memory index on strain JBC5 over control strain by CI = +0.44 (***p < 0.001) (Figure. 4 B and C).
C. elegans were pre-cultured on JBC5 or control for 3 days and transferred to nematode growth media (NGM) plates seeded with pathogen S. aureus and their survival was analyzed. The pre-culturing of C. elegans on strain JBC5 increased their survival against pathogen S. aureus by 25% compared control-fed (**p < 0.01) (Figure. 4D).
To further study the effect of strain JBC5 on intestinal integrity of C. elegans, adult C. elegans were cultured on strain JBC5 or control for 5 days and then transferred onto NGM plates seeded with pathogen S. aureus. C. elegans were then grown in a liquid NGM medium containing heat-killed E. coli OP50 stained with blue food dye to observe the intestinal integrity. It was observed that the C. elegans pre-cultured on strain JBC5 has lesser distention of intestinal lumen against pathogenic compared to fed with control bacterium (Mean ± SEM, 20.8 ± 1.38 for JBC5 vs 38.26 +2.07 on control bacterium) (**p < 0.01) (Figure. 4 E and F).
Figure. 4 A. Schematic diagram showing the experimental design. The experiment was performed on a 60 mm Petri-plate containing NGM medium for the food preference assay. For food preference assay, 3-days old C. elegans were transferred onto the center of the plates equidistant from both the bacterial lawns and counted after 4 hours of incubation at 20 °C. For training, the C. elegans were transferred to NGM plates seeded with LPJBC5 for 4 hours and kept at 20 °C. B and C.
The binary food choice assay was performed as Choice index (CI) was calculated for C. elegans. Memory index (CI trained- CI naive) was calculated from the results of the binary choice assay (Scale bar, 1 mm). Error bars represent mean ± SEM. CI = -1.0 shows complete food preference for control food OP50. CI = + 1.0 shows complete food preference for testing bacteria JBC5. CI = 0.0 shows equal distribution of food preference for both OP50 or JBC5. D. Survival of C. elegans pre-cultured on JBC5 or control for 3 days and then infection was given on pathogen Staphylococcus aureus and maintained at 20 °C.
E and F Feeding of JBC5 improved intestinal integrity of C. elegans against pathogen S. aureus. The intestinal cavity was observed in treated groups under a compound microscope at 20X (Scale bar, 20 pm). Error bars represent ± SEM and each group were statistically analyzed using Student's t-test (*p < 0.05, **p < 0.01 and ***p < 0.001).
Further, strain JBC5-induced longevity mechanisms in C. elegans was studied. Previous studies suggested that mainly two molecular pathways operate in order to extend longevity, i.e. p38 MAPK and DAF-2/DAF-16 pathways (Kumar et al. 2020). Our studies on the mutants of C. elegans suggested that strain JBC5 were able to extend longevity in loss-of-function daf-2 (short-lived) and daf-16 (long- lived) mutants (daf-2, ***p < 0.0001; daf-16, ***p < 0.0001 by the log-rank test) (Data not shown). We observed no extension in the longevity of C. elegans gene mutant involved in p38 MAPK pathway, namely pmk-1, sek-1 and nsy-1 (p > 0.05 by the log-rank test) (Data not shown). In support, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results showed increase in the
expression of genes involved in p38 MAPK signaling (pmk-1, sek-1 and nsy-1), while there was no significant change in the expression of daf-2 and daf-16 (Figure. 5a).
Previous research showed that downstream gene of p38 MAPK signaling, namely skn-1 has been found to regulate expression of anti-oxidative genes involved in phase-2 detoxification. It was observed that feeding of LPJBC5 showed no extension in the longevity of skn-1 mutant (p > 0.05 by the log-rank test) (Data not shown). We further found that expression of skn-1 gene was increased by two-fold in JBC5-fed C. elegans (Figure. 5a). Next, it was also found that the increase in the expression of transcription factor SKN-1 upregulates the expression of anti-oxidative genes, namely catalases (ctl-1 and ctl-2), trx-1, GSTs (gst-4 and gst-7) and SODs (sod-1 and sod-3) (Figure. 5a).
The reduced expression of fat related genes was also found, namely fat-5 and fat-7 in JBC5-fed C. elegans (Figure. 5a). Serotonin signaling has been found in regulating the learning and memory of C. elegans (Tsui and van der Kooy 2008). Therefore, further, the expression of genes involved in serotonin signaling were studied, namely mod-1, ser-1 , and tph-1. The expression of all three serotonin signaling genes were found to be significantly high (Figure. 5a).
In this study, adult C. elegans were pre-cultured on strain JBC5 or control for and exposed to gut pathogen S. aureus. It was found that strain JBC5 improved the survival of C. elegans against infection of pathogen S. aureus as well as improved intestinal integrity of C. elegans (Figure 4 D-F). Furthermore, the expression of innate immunity genes were found to be significantly higher in JBC5-fed C. elegans, namely lysozymes (lys-1 and lys-8), antibacterial factor (ABF) (abf-2 and abf-3), saponin-like proteins (spp-7) and C-type lectin (CLEC) domain containing proteins (clec-60 and clec-85) (Figure. 5b). In addition, two-fold increase in the expression of tight junction protein, zonula occludin zoo-1 in JBC5-fed C. elegans was also observed. (***p < 0.001) (Figure. 5b).
Accompanying figure 5a illustrates qRT-PCR analysis on the mRNA expression of genes involved in longevity, anti-oxidation, fat accumulation, and learning and memory. Figure 5b illustrates. mRNA expression of genes involved in innate immunity and tight junction proteins (ZOO-1) in maintaining intestinal integrity against infection with pathogen S. aureus. mRNA expression was normalized using the house-keeping gene act-1. Error bars represent ± SEM and each group were statistically analyzed using Student's t-test (*p < 0.05, **p < 0.01 and ***p < 0.001).
In conclusion, the results suggest that strain JBC5 activates p38 MAPK signaling cascade in C. elegans which further activates its downstream gene skn-1 involved in phase-II detoxification, anti-pathogenic genes and intestinal integrity. Feeding of strain JBC5 reduced the expression of fat accumulation genes and increased the expression of serotonin signaling genes involved in learning and memory (Figure 6).
Accompanying figure 6 illustrate predicted mechanisms of JBC5 induced antiaging mechanisms in C. elegans.
Feeding of strain JBC5 activates p38 MAPK signaling cascade which further activates the downstream SKN-1 transcription factor involved in phase-II detoxification or antioxidation. These higher expressions of antioxidative genes promoted longevity, increased the expression of genes involved in stress resistance and infection against pathogen S. aureus. The feeding of strain JBC5 also reduces the expression of fat-related genes as well as improve the expression of serotonin-signaling genes involved in learning and memory. In result, the feeding of strain JBC5 extends longevity as well as promote reduces other age-associated pathologies in C. elegans.
From the above results and elaborate description, it is clear that the present invention provides: a) method for production of functional curd with anti-ageing property, consistent curdling and high shelf life.
b) preparing cowmilk curd with defined proportions of starter inoculum including Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 having anti-ageing and curdling property. c) bacterial strain L. plantarum JBC5 possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
From the description hereinbefore it would be clear that the method is a simple and fast as well as precise. Cost of production is reasonably low. The present invention has been described with reference to some drawings, test results and preferred embodiments, purely for the sake of understanding and not by way of any limitation and the present invention includes all legitimate developments within the scope of what has been described hereinbefore and claimed in the appended claims.
Claims
1. A method for preparation of functional curd with anti-ageing property and high shelf life comprising: a) collecting fresh cow milk from a dairy farm aseptically in a sterilized bag, b) maintaining refrigerated condition (4°C), c) pasteurizing at 100°C for 2 minutes and allowing it to cool, d) fermenting the said milk with a starter culture comprising Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 such that the ratio of Lactobacillus plantarum JBC5: Lactobacillus, brevis H4 = 109cfu/ml in 1 : 1 concentration and obtaining the desired functional curd.
2. The method as claimed in claim 1, wherein Lactobacillus plantarum JBC5 is isolated from a local variety of yoghurt.
3. The method as claimed in claim 1, wherein Lactobacillus brevis H4 is isolated from honey.
4. The method as claimed in claim 1, wherein the starter culture is optimized using different colony forming units and selecting the one which stabilized Lactobacillus plantarum JBC5 and increased the shelf life, anti-ageing property and consistent curdling of functional curd.
5. A functional curd obtained by the method as claimed in claims 1 to 4.
6. A starter inoculum for preparing cow milk curd having anti-ageing, high shelf and excellent curdling properties comprising Lactobacillus plantarum JBC5 and Lactobacillus brevis H4 having anti-ageing and curdling property wherein the ratio of Lactobacillus plantarum JBC5: Lactobacillus, brevis H4 = 109cfu/ml in 1 : 1.
7. The starter inoculum as claimed in claim 6, wherein the bacterial strain L. plantarum JBC5 possesses probiotic and diverse features of anti-ageing including gut integrity and cognitive functions which is unique.
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| WEERATHILAKE W A D V, RASIKA, RUWANMALI J K U, MUNASINGHE M A D D: "The evolution, processing, varieties and health benefits of yogurt", INTERNATIONAL JOURNAL OF SCIENTIFIC AND RESEARCH PUBLICATIONS, INTERNATIONAL JOURNAL OF SCIENTIFIC AND RESEARCH PUBLICATIONS, vol. 4, no. 4, 1 April 2014 (2014-04-01), XP093080647, ISSN: 2250-3153 * |
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| CN114480407B (en) * | 2021-12-07 | 2023-10-20 | 南京安吉生物科技有限公司 | Clec126 gene in caenorhabditis elegans genome and application thereof |
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