AU2020103508A4

AU2020103508A4 - Probiotic Blend

Info

Publication number: AU2020103508A4
Application number: AU2020103508A
Authority: AU
Inventors: Peter Strover
Original assignee: Probiotic Co Pty Ltd
Current assignee: Probiotic Co Pty Ltd
Priority date: 2019-11-20
Filing date: 2020-11-18
Publication date: 2021-01-28
Anticipated expiration: 2028-11-18

Abstract

The application relates to a probiotic blend for use in a substantially unfermented beverage product. According to certain embodiments, the probiotic blend may provide for suitable probiotic survivability under refrigerated conditions without fermenting the beverage product or otherwise negatively affecting taste. According to certain embodiments, the beverage product may be milk-based, optionally cow milk-based.

Description

PROBIOTIC BLEND FIELD

[0001] The present invention is generally directed a probiotic blend and a food or beverage product comprising a probiotic blend.

BACKGROUND

[0002] Probiotics, broadly defined as good or helpful live yeast or bacteria, form a part of many traditional food products, including fermented dairy products such as yoghurt and kefir, fermented vegetable products such as sauerkraut and kim chi, and fermented drink products such as kombucha.

[0003] In 1907, nobel laureate lie Metchnikoff postulated that probiotics may improve health by improving gut flora. Metchnikoff observed that rural populations of areas of Europe such as Bulgaria, whose diets consisted largely of milk fermented by lactic-acid bacteria, enjoyed unusually long lifespans. Research has since indicated that probiotics may support digestive health and immune function, including reducing antibiotic-associated diarrhoea, improving resilience to infections, and improving digestion of lactose. Other claimed benefits of probiotics include reducing the risk of eczema and colic in infants.

[0004] Incorporation of live probiotic microorganisms into food and beverage products has become more prevalent as consumers seek those health benefits exemplified above. Since probiotics are understood as only effective while live, any application of probiotics should ensure that sufficient live microbes remain at the time of intake, and it is often necessary to increase the amount of live culture several-fold during manufacture to account for survival rates prior to eventual consumption. In 2002, the World Health Organisation and the Federal Drugs Administration (US) recommended that'the minimum viable numbers of each probiotic strain at the end of shelf life be reported' on labelling. However, suppliers generally continue to report viable numbers on manufacture, since it the prediction of viable numbers at the end of shelf life would depend on transport and storage conditions.

[0005] It goes without saying that one must consume probiotics before obtaining any health benefits from them. In this respect, consumer products containing probiotics should not compromise sensory aspects such as smell and taste. Research has previously shown that most consumers remain unwilling to compromise the flavour of probiotic products to obtain any health benefits. This is particularly problematic since the metabolism of probiotic bacteria can produce compounds having a characteristic 'probiotic off flavour', caused at least in part by fermentation / culturing.

[0006] WO patent publication 2008/117302 Al (the entire disclosure of which is hereby incorporated) discloses a process for the preparation of a probiotic fermented milk beverage with enhanced stability and metal ions with adjuvant supplementation. The disclosed process comprises: inoculating a starter culture comprising a Leuconostoc mesenteroides sub-species having MTCC NO 5209 followed by incubation at about 37°C for about 24 hours; adding to the starter culture adjuvants selected from the group consisting of tryptone, casein hydrolysate, cysteine hydrochloride and Ascorbic acid at a concentration between 50 500mg/mL. While the disclosed process is said to provide increased probiotic stability, it is reliant on a process which necessitates: provision of a fermented milk product, specific use of Leuconostoc mesenteroides as a probiotic, and addition of adjuvants to increase the concentration of trace metal ions within the resultant fermented milk product.

[0007] EP patent publication 2194118 Al (the entire disclosure of which is hereby incorporated) discloses a method of increasing the survivability of lactic acid bacterium in fermented milk products such as yoghurt. According to EP 2194118, the survival rate of lactic acid bacterial strains may be improved by incorporating a fermentation product of a propionic acid bacterium into a suitable food or beverage product. Suitable Propionibacteria for to provide such fermentation products include bacteria for cheese such as: Propionibacterium freudenreichii, Propionibacterium jthoenii, Propionibacterium acidipropionici, and Propionibacteriumjensenii. The propionic acid bacterium can be cultured in a medium derived predominantly from whey decomposed with Protease Amano. According to EP 2194118 the resulting fermentation product may improve the survival rate of lactic acid bacteria in low-pH environments, such as those found in fermented milk products (e.g. pH of 3.8-4.8). Microorganisms with confirmed improved survivability include strains of: Lactobacillus gasseri, Lactobacillus bulgaricaus, and Streptococcus thermophilus. The disclosed process exclusively effective in low pH environments, and appears to necessitate a separate fermentation process to provide the necessary fermentation products of a propionic acid bacterium. Further, EP 2194118 is limited to use of probiotics in unfermented products.

[0008] EP patent publication 2605669 Al (the entire disclosure of which is herein incorporated) discloses a method of improving the survival rate of a probiotic strain by combining the probiotic strain with a Lactobacillus strain that improves the survival rate of the probiotic strain. In certain embodiments, the probiotic strain may be selected from either Lactobacillus acidophilus or Bifidobacterium animalis lactis, and the Lactobacillus strain which improves the survival rate of a probiotic strain may be a Lactobacillus strain such as

Lactobacillus rhamnosus or Lactobacillus paracasei, consisting specifically of Lb2132, Lbc 82, LC-10, Lpc-37 and Lr-32. EP 2605669 teaches that the ratio of probiotic strain versus Lactobacillus strain to improve survival rate of the probiotic strain may be quite broad, for example 1:10 to 50:1. In this respect the rate of combination of these strains is not taught as particularly essential. EP 2605669 is particularly directed toward production of a fermented milk drink in which an inoculum of the combination of strains is combined with milk and a starter culture, and the milk is fermented under known suitable conditions. EP 2605669 is not directed toward selection of a probiotic product suitable for use in unfermented milk products so as to maintain a suitable sensory profile, for example.

[0009] WO patent publication 2010/023290 A2 (the entire disclosure of which is herein incorporated) discloses a method for improving the growth of Bifidobacteria by incorporation of selected bacterial strains in fermented milk products. In particular, WO 2010/023290 teaches that the addition of specific Lactococcus lactis strains significantly improves the growth of Bifidobacteria during fermentation of the milk such that large amounts of Bifidobacteria are available in a final fermented milk product. By using strains of Lactococcus lactis, a fermented milk product containing more than 108 cfu/g Bifidobacteria can be created directly from fermentation. Additionally, WO 2010/0232290 A2 teaches that addition of a specific strain of Streptococcus thermophilus (CHCC7018) may also improve the growth of Bifidobacteria. WO 2010/023290 solves the problem of providing high probiotic availability by ensuring high Bifidobacteria growth during fermentation of a fermented milk product. WO 2010/023290 is not directed to improving the longevity of probiotic cells per se or toward use of probiotics in unfermented beverage products.

[0010] Like WO 2010/023290, WO patent publication 2008/148561 Al (the entire disclosure of which is herein incorporated) discloses a method for improving growth of Bifidobacteria by incorporation of a selected bacterial strain in fermented milk products. WO 2008/148561 specifically teaches that use of a specific Stroptococcus thermophilus bacteria (ST6008) may improve growth of Bifidobacteria during fermentation. Like WO 2010/023290, WO 2008/148561 teaches a method of improving probioatic availability by improving the growth of a probiotic during fermentation of a milk product (i.e. to improve the number of cells available immediately following fermentation). WO 2008/148561 is not directed to improving the longevity of probiotic cells per se or toward use of probiotics in unfermented products.

[0011] Despite previous advances such as those exemplified above, it would be desirable to develop new applications for probiotics in food and beverage products so as to broaden their appeal, and it would be particularly desirable to provide new application in a manner which provides for suitable shelf life.

[0012] It is an object of the invention to provide a probiotic which overcomes one of more of disadvantages of the above prior art.

[0013] The above discussion of background art is included to explain the context of the present invention. It is not to be taken as an admission that the background art was known or part of the common general knowledge at the priority date of any one of the claims of the specification.

SUMMARY

[0014] A probiotic blend suitable for inclusion in a substantially unfermented beverage product, the probiotic blend comprising: Lactobacillus acidophilus, Bifidobacterium animalis lactis, and Lactobacillus rhamnosis.

[0015] Optionally, the probiotic blend comprises Lactobacillus acidophilus, Bifidobacterium animalis lactis, and Lactobacillus rhamnosis in the following respective proportions (by CFU): 0.5-2.0 : 0.5-2.0 : 0.5-2.0, further optionally: 0.8-1.25 : 0.8-1.25 : 0.8-1.25, further optionally substantially 1 : 1: 1.

[0016] Optionally, the probiotic blend comprises: 23-43 % Lactobacillis acidophilus, 25-45% Bifidobacterium animalis lactis, and 22-42% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

[0017] Optionally, the probiotic blend comprises: 28-38 % Lactobacillis acidophilus, 30-40% Bifidobacterium animalis lactis, and 27-37% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

[0018] Optionally, the probiotic blend comprises: 31-35 % Lactobacillis acidophilus, 33-37% Bifidobacterium animalis lactis, and 30-34% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

[0019] Optionally, the probiotic blend comprises: substantially 33% Lactobacillis acidophilus, substantially 35% Bifidobacterium animalis lactis, and substantially 32% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

[0020] Optionally, the probiotic blend substantially consists of: Lactobacillus acidophilus, Bifidobacterium animalis lactis, and Lactobacillus rhamnosis.

[0021] Optionally, the probiotic blend is configured to ensure suitable survivability of probiotics in a beverage product under refrigerated conditions without substantially fermenting the beverage product.

[0022] According to a second aspect of the invention there is provided a substantially unfermented beverage product comprising a probiotic blend according to a first aspect of the invention.

[0023] Optionally, the beverage comprises as a major component a base product selected from: fruit juice, vegetable juice, plant milk, or animal milk.

[0024] Optionally, the base product is plant milk.

[0025] Optionally, the base product is: almond milk, coconut milk, oat milk, soy milk, rice milk, or mixtures thereof.

[0026] Optionally, the base product is animal milk.

[0027] Optionally, the base product is: cow milk, sheep milk, goat milk, camel milk, buffalo milk, or mixtures thereof.

[0028] Optionally, the beverage product comprises between 10-50 billion CFU per litre of the probiotic blend immediately upon production

[0029] Optionally, the beverage product comprises between 15-40 billion CFU per litre of the probiotic blend immediately upon production.

[0030] Optionally, the beverage product comprises between 20-30 billion CFU per litre of the probiotic blend immediately upon production.

[0031] Optionally, the beverage product contains at least 3 billion CFU, preferably 5 billion CFU, of probiotic blend after 14 days in refrigerated conditions.

[0032] Optionally, the beverage product comprises (immediately upon production): 5-10 billion CFU per litre of Lactobacillis acidophilus; 5-10 billion CFU per litre of Bifidobacterium animalis lactis, and 5-10 billion CFU per litre of Lactobacillus rhamnosis.

[0033] Optionally, the beverage product comprises (immediately upon production): substantially 8.25 billion CFU per litre of Lactobacillis acidophilus; substantially 8.75 billion CFU per litre of Bifidobacterium animalis lactis, and substantially 8.0 billion CFU per litre of Lactobacillus rhamnosis.

[0034] According to a third aspect of the invention there is provided a method of producing a substantially unfermented beverage product according a second aspect of the invention, the method comprising separately adding: Lactobacillus acidophilus, Bifidobacterium animalis lactis, and Lactobacillus rhamnosis to beverage product comprising a base product selected from: fruit juice, vegetable juice, plant milk or animal milk.

[0035] Optionally, the method comprises adding a premixed blend of: Lactobacillus acidophilus, Bifidobacterium animalis lactis, and Lactobacillus rhamnosis to a beverage product comprising a base product selected from: fruit juice, vegetable juice, plant milk or animal milk.

[0036] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations thereof such as "comprises" and "comprising", will be understood to include the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or groups of integers or steps.

DETAILED DESCRIPTION

[0037] It will be convenient to further describe embodiments of the invention, as well as research relating to the invention, with reference to the accompanying drawings. Other embodiments are possible, and consequently, the particularity of the accompanying drawings is not to be understood as superseding the generality of the preceding description of the invention.

[0038] Experiments were conducted with the aim of advantageously developing a probiotic blend that could be used in a beverage product so as to substantially avoid affecting taste and pH, to provided suitable shelf life with suitable survivability of probiotics, and to substantially avoid fermenting the resulting beverage product. In this context, a substantially unfermented product includes products that are for all relevant intents and purposes unfermented without necessarily completely avoiding all fermentation reactions.

[0039] As part of the experiments, mixtures of probiotics and full-cream milk (having a 'best before' period of at least 10 days) were developed and evaluated. Samples for testing were generally prepared according to scientific procedures, such as that exemplified below:

2L of full-cream pasteurised and homogenised milk is poured into a 5L sterile scotch bottle including a magnetic stirrer;

with the milk gently stirring, a pre-weighed amount of probiotics is added and the mixture is stirred for 8-10 minutes;

14L of further milk is added to a separate 19L sterile pot including a magnetic stirrer; the 2L milk/probiotic mixture is slowly added while the 14L of further milk is gently stirred, and stirring continues for a further 8-10 minutes to achieve a uniform blend; the 16L of milk probiotic mixture is then pumped into 2L milk containers for storage under refrigerated conditions.

[0040] Samples were tested at various stages of the consumer life cycle. Generally, samples were tested: on initial mixing, after 3 days, after 7 days and after 14 days. Occasionally, samples were further tested after 21 days of refrigerated storage (i.e. at or below 4C). Testing included: organoleptic testing (i.e. to test taste and smell), pH (to test for lactic acid production), CFU concentrations (i.e. to test survivability of probiotics).

[0041] The inventors initially tested several potentially suitable probiotic strains, but identified issues in respect of unsatisfactory oxidation (fermentation or culturing) of milk during shelf life. In many instances, the resulting probiotic blend product either: provided a 'probiotic flavour', developed a highly undesirable fermented/acidic flavour profile, or demonstrated insufficient survivability of probiotics during shelf life (such that insufficient probiotics were available for intake following typical periods and conditions of refrigerated storage).

[0042] Following initial testing of several probiotic strains, the inventors eventually identified L acidophilus, L rhamnosis, and B animalis lactis as suitable probiotics for further consideration, and experiments were then performed to consider how each of these probiotic strains performed in isolation in milk at different initial concentrations as set out in the below Table 1.

Table 1 Concentration (Billion CFU/ L) Survival(%) Initial Day 3 Day 7 Day 14 Day 7 Day 14 1 L acidophilus 287 182 156 172 54 60 2 L acidophilus 260 177 156 9.6 60 3.7 3 B animalis lactis 54 23.5 18.8 0 35 0 4 B animalis lactis 60 30.5 12.5 1.5 20 2.5 L rhamnosis 1120 800 643 5.9 57 0.5 6 L rhamnosis 390 355 383 1.95 98 0.5

[0043] The used strains of: L acidophilus, L rhamnosis, and B animalis lactis were supplied by 'Probiotics Australia' (Australian Business Number: 36 135 032 138) and are identified by Probiotics Australia as follows:

Table 2

Description Strain ID L acidophilus PA-LA01 L rhamnosis PA-LR12 B animalis subspecies lactis PA-BL21

[0044] With the exception of sample 1 of Table 1, the survival rate of probiotics was generally below 4% after 14 days, indicating that large initial dosages of probiotics were required to ensure sufficient survival of probiotics after 14 days. B animalislactic also demonstrated lower survival rates than L acidophilus and L rhamnosis after 7 days.

[0045] In addition to survivability analysis as demonstrated above, samples from Table were assessed for pH, odour and taste. Test results were as set out in Table 3 below.

Table 3

Organoleptic scores pH Day 3 Day 7 Day 14 Initial Day 3 Day 7 Day 14 1 &2 L acidophilus 8.5-9.0 8.5-9.0 8.0 6.72 6.72 6.71 6.74 3 &4 B animalis lactis 8.5-9.0 8.0-9.0 7.5-8.5 6.72 6.72 6.70 6.70 L rhamnosis 6.0 5.5 3.5 6.72 6.63 6.64 6.64 6 L rhamnosis 9.0 9.0 9.0 6.72 6.71 6.72 6.67 7 full-cream milk 9.0 8.5 8.5 6.72 6.71 6.71 6.7

[0046] Generally speaking, with the exception of sample 5, which include a comparatively large concentration of probiotics, addition of the probiotics to the milk samples at the provided concentrations did not negatively affect organoleptic scores or pH. The concentration of L rhamnosis in sample 5 was considered so high as to render it unsuitable for consumer acceptance.

[0047] Based on the above results, several blends incorporating L acidophilus, B animalis lactis, and L rhamnosis were proposed. In particular, a blend as set out below in Table 4 was tested.

Table 4

Initial Conditions Relative proportions Billion CFU / L L acidophilus 33% 8.25 B animalis lactis 35% 8.75 L rhamnosis 32% 8

[0048] Following production, two milk blend samples were tested over 14 days against a pure milk control sample. The results were as set in Table 5 below.

Table 5

Organoleptic scores pH Conc. (Billion CFU / L) Day 0 Day 7 Day 14 Day 0 Day 7 Day 14 Day 0 Day 7 Day14 Control 9.5 9.5 8.5 6.55 6.58 6.50 - - Sample 1 9 9.5 9 6.56 6.58 6.55 23.0 30.2 8.5 Sample 2 8.5 10 9 6.61 6.63 6.57 27.4 10.7 6.6

[0049] The results showed a probiotic blend which provided organoleptic scores comparable to that of the control sample over 14 days. Similarly, results showed that the probiotic blends did not negatively affect pH, indicating that the probiotic blend did not cause lactic acid production (i.e. bacterial fermentation). Lastly, the results indicated suitable survivability of the probiotics over a 14 day period in which about 25-37% of probiotics remained after 14 days (under refrigerated conditions), an increase over the survivability generally indicated by Table 1. It is also noted that Samples 1 and 2 provided suitably high survivability after 21 days (12% or better).

[0050] The inventors have since performed further tests which have demonstrated survivability consistent with the above, without fermenting the milk product or otherwise affecting organoleptic scores.

[0051] While the testing described above was performed utilising a cow milk base, other suitable base products are contemplated according to the invention, including: plant milk such as almond milk, coconut milk, oat milk, soy milk, rice milk, or mixtures thereof and other animal milks including sheep milk, goat milk, camel milk, buffalo milk, or mixtures thereof. Use of skim or low-fat milks is further contemplated.

[0052] In addition, while testing was performed using pure milk, additional materials may be added to the beverage product as may be understood in the art. Flavourings or other additives may be added to the beverage product such as chocolate (to for example provide chocolate milk), caramel, strawberry and others.

[0053] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations thereof such as "comprises" and "comprising", will be understood to include the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or groups of integers or steps.

[0054] It will be understood to persons skilled in the art of the invention that modifications may be made without departing from the spirit and scope of the invention. The embodiments and/or examples as described herein are therefore to be considered as illustrative and not restrictive.

Claims

1. A probiotic blend suitable for inclusion in a substantially unfermented beverage product, the probiotic blend comprising: Lactobacillus acidophilus, Bifidobacterium animals lactis, and Lactobacillus rhamnosis.

2. A probiotic blend according to claim 1, wherein the probiotic blend comprises Lactobacillus acidophilus, Bifidobacterium animals lactis, and Lactobacillus rhamnosis in the following respective proportions (by CFU): 0.5-2.0 : 0.5-2.0 : 0.5-2.0, further optionally: 0.8-1.25 : 0.8-1.25 : 0.8-1.25, further optionally substantially 1 : 1: 1.

3. A probiotic blend according to claim 2, wherein the probiotic blend comprises: 23-43 % Lactobacillis acidophilus, 25-45% Bifidobacterium animals lactis, and 22-42% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

4. A probiotic blend according to claim 3, wherein the probiotic blend comprises: 28-38 % Lactobacillis acidophilus, 30-40% Bifidobacterium animals lactis, and 27-37% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

5. A probiotic blend according to claim 4, wherein the probiotic blend comprises: 31-35% Lactobacillis acidophilus, 33-37% Bifidobacterium animals lactis, and 30-34% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

6. A probiotic blend according to claim 5, wherein the probiotic blend comprises: substantially 33% Lactobacillis acidophilus, substantially 35% Bifidobacterium animals lactis, and substantially 32% Lactobacillus rhamnosis (calculated against total CFU of the three listed probiotics).

7. A probiotic blend according to any one of the previous claims, wherein the probiotic blend substantially consists of: Lactobacillus acidophilus, Bifidobacterium animals lactis, and Lactobacillus rhamnosis.

8. A probiotic blend according to any one of the previous claims wherein the probiotic blend is configured to ensure suitable survivability of probiotics in a beverage product under refrigerated conditions without substantially fermenting the beverage product.

9. A substantially unfermented beverage product comprising a probiotic blend according to any one of the previous claims.

10. A beverage product according to claim 9, wherein the beverage comprises as a major component a base product selected from: fruit juice, vegetable juice, plant milk, or animal milk.

11. A beverage product according to claim 10, wherein the base product is plant milk.

12. A beverage product according to claim 11, wherein the base product is: almond milk, coconut milk, oat milk, soy milk, rice milk, or mixtures thereof.

13. A beverage product according to claim 10, wherein the base product is animal milk.

14. A beverage product according to claim 13, wherein the base product is: cow milk, sheep milk, goat milk, camel milk, buffalo milk, or mixtures thereof.

15. A beverage product according to any one of claims 9 to 14, wherein the beverage product comprises between 10-50 billion CFU per litre of the probiotic blend immediately upon production

16. A beverage product according to claim 15, wherein the beverage product comprises between 15-40 billion CFU per litre of the probiotic blend immediately upon production.

17. A beverage product according to claim 16, wherein the beverage product comprises between 20-30 billion CFU per litre of the probiotic blend immediately upon production.

18. A beverage product according to any one of claims 9 to 17, wherein the beverage product contains at least 3 billion CFU, preferably 5 billion CFU, of probiotic blend after 14 days in refrigerated conditions.

19. A beverage product according to any one of claims 9 to 18, wherein the beverage product comprises (immediately upon production): 5-10 billion CFU per litre of Lactobacillis acidophilus; 5-10 billion CFU per litre of Bifidobacterium animalis lactis, and 5-10 billion CFU per litre of Lactobacillus rhamnosis.

20. A beverage product according to any one of claims 9 to 19, wherein the beverage product comprises (immediately upon production): substantially 8.25 billion CFU per litre of Lactobacillis acidophilus; substantially 8.75 billion CFU per litre of Bifidobacterium animalis lactis, and substantially 8.0 billion CFU per litre of Lactobacillus rhamnosis.

21. A method of producing a substantially unfermented beverage product according to any one of claims 9 to 20, themethod comprising separately adding: Lactobacillus acidophilus, Bifidobacterium animalis lactis, and Lactobacillus rhamnosis to beverage product comprising as a major component a base product selected from: fruit juice, vegetable juice, plant milk or animal milk.

22. A method of producing a substantially unfermented beverage product according to any one of claims 9 to 20, the method comprising adding a premixed blend of: Lactobacillus acidophilus, Bifidobacterium animalis lactis, and Lactobacillus rhamnosis to a beverage product comprising as a major component a base product selected from: fruit juice, vegetable juice, plant milk or animal milk.